Use of Lyso-GB1 as Druggable Target

ABSTRACT

The present invention is related to the in vitro use of lyso-Gb1 as a draggable target in the development of a drug, and to antagonist of lyso-Gb1 for use in the treatment and/or prevention of a disease, wherein the disease is Gaucher disease or Parkinson&#39;s disease

The present invention is related to the use of lyso-Gb1, an antagonistof lyso-Gb1, use of an antagonist of lyso-Gb1, a method for thegeneration of an animal model for a disease, a method for the screeningof an agent suitable for and/or capable of treating and/or preventing adisease, and a method for the assessment of the effects of an agent inthe treatment and/or prevention of a disease.

Modern drug development no longer relies on a more or less heuristicapproach, but typically involves the elucidation of the molecularmechanism underlying a disease or a condition, the identification ofcandidate target molecules and the evaluation of said target molecules.Once such a validated target molecule, which is herein referred to alsoas target, is available, drug candidates directed thereto may be tested.In many cases such drug candidates are members of a compound librarywhich may consist of synthetic or natural compounds. Also the use ofcombinatorial libraries is common. Such compound libraries are hereinalso referred to as candidate compound libraries. Although in the pastthis approach has proven to be successful, it is still time and moneyconsuming. Different technologies are currently applied for targetidentification and target validation.

A further prerequisite for drug development is the availability of ananimal model for a disease or condition in question. The absence of anappropriate animal model increases the risk of failure of any drugdevelopment as there is a considerable gap between cell-based assaysused in the screening, characterization and assessment of drugcandidates and the testing of such drug candidates in a human body, withthe results obtained in cell-based assays typically not allowing acomprehensive and fact-based prediction as to the behaviour of a drugcandidate in a human body. However, also the quality of an animal modelfor a disease and condition, respectively, in question is important. Thebetter the animal model reflects the particularities of the disease anddisorder, respectively, in question in man, the more reliable are theresults obtained from the testing of a drug candidate whichsignificantly reduces the risk that a drug candidate ultimately fails inthe treatment of a human patient. Among the plethora of human diseases,lysosomal storage diseases, also referred to herein as lysosomal storagedisorders or LSDs, are a group of rare inherited metabolic disordersthat result from defects in lysosomal function. LSDs result when aspecific organelle in the body's cells—the lysosome—malfunctions. Someof the more prominent lysosomal storage diseases are Gaucher's diseaseand Fabry disease.

LSDs are caused by lysosomal dysfunction usually as a consequence ofdeficiency of a single enzyme required for the metabolism of lipids,glycoproteins or so-called mucopolysaccharides. Individually, LSDs occurwith frequencies of about 1:10,000 to 1:250,000, however, as a group theincidence is about 1:5,000. Most of these disorders are autosomalrecessively inherited; however, a few are X-linked inherited, such asFabry disease and Hunter syndrome.

Like other genetic diseases, individuals typically inherit lysosomalstorage diseases from their parents. Although each disorder results fromdifferent gene mutations that translate into a deficiency in enzymeactivity, they all share a common biochemical characteristic—nearly alllysosomal disorders originate from an abnormal accumulation ofsubstances inside the lysosome.

Lysosomal storage diseases affect mostly children and they often die atyoung and unpredictable age, many within a few months or years of birth.Many other children die of this disease following years of sufferingfrom various symptoms of their particular disorder.

The symptoms of lysosomal storage disease vary, depending on theparticular disorder and other variables like the age of onset, and canbe mild to severe. They can include developmental delay, movementdisorders, seizures, dementia, deafness and/or blindness. Some peoplewith lysosomal storage disease have enlarged livers (hepatomegaly) andenlarged spleens (splenomegaly), pulmonary and cardiac problems, andbones that develop abnormally.

So far, there are no causative cures for lysosomal storage diseases andtreatment is mostly symptomatic, although bone marrow transplantationand enzyme replacement therapy (abbr. ERT) have been used for someindications with good success. In addition, umbilical cord bloodtransplantation is being performed at specialized centers for a numberof these diseases. In addition, substrate reduction therapy (abbr. SRT),a method used to decrease the accumulation of storage material, iscurrently being evaluated for some of these diseases. Furthermore,chaperone therapy, a technique used to stabilize the defective enzymesproduced by patients, is being examined for certain of these disorders.Gene therapy constitutes a further option for the treatment of thesediseases.

One prominent disease from the group of lysosomal storage diseases isGaucher disease. Gaucher disease is caused by a genetic defect of theglucocerebrosidase gene (abbr. GBA gene) resulting in a deficiency ofthe corresponding lysosomal enzyme glucocerebrosidase (abbr. GCase). Thedeveloping discrepancy of glucosylceramide (abbr. GlcCer) build-up inthe Endoplasmic Reticulum and degradation in the lysosomes of the cellsleads to prominent accumulation of GlcCer in tissue macrophages. Amongthe major clinical symptoms of Gaucher disease are liver and spleenenlargement, decreased red blood cells and thrombocytes and skeletalabnormalities causing an increased risk of fractures and osteonecrosis.Glucosylsphingosine (abbr. lyso-Gb1), the deacylated form ofglucosylceramide, has recently been identified as a sensitive andspecific biomarker for Gaucher disease (see, for example, internationalpatent application WO 2012/167925).

The accumulation of the lyso-Gb in cerebrum and cerebellum in infantileand juvenile GD patients has firstly been documented in 1982 (Nilsson etal., 1982, J Neurochem 39: 709-718). Lyso-Gb1 is an amphipathic compoundthat has been reported to originate from the enzymatic action oflysosomal acid ceramidase on the cell's primary glycosphingolipidstorage product, glucosylceramide (Flanagan et al., 2013, Mol GenetMetab 108:S40-41; Ferraz et al., 2016, FEBS Lett 590:716-725). Lyso-Gb1is highly abundant in the brain tissue of patients with neuronopathicGD, but not in non-neuronopathic GD patients (Orvisky et al, 2002, MolGenet Metab 76: 262-70).

A problem underlying the present invention was the provision of a targetwhich is suitable for therapeutic approaches in the treatment of alysosomal storage disorder and Gaucher disease in particular.

A problem underlying the present invention was the provision of a targetwhich is suitable for therapeutic approaches in the treatment ofParkinson's disease.

Another problem underlying the present invention was the provision of ananimal model, more particularly an animal model for a lysosomal storagedisorder and Gaucher disease in particular, and means for the generationof such animal model.

Another problem underlying the present invention was the provision of ananimal model, more particularly an animal model for Parkinson's disease,and means for the generation of such animal model.

A further problem underlying the present invention was the provision ofmeans for the treatment and/or prevention of a disease, preferably alysosomal storage disorder and more preferably Gaucher disease.

A further problem underlying the present invention was the provision ofmeans for the treatment and/or prevention of a disease, preferablyParkinson's disease.

A still further problem underlying the present invention was theprovision of a method for the screening of an agent suitable for and/orcapable of treating and/or preventing a disease, whereby by disease ispreferably a lysosomal storage disorder and more preferably Gaucherdisease.

A still further problem underlying the present invention was theprovision of a method for the screening of an agent suitable for and/orcapable of treating and/or preventing a disease, whereby by disease isParkinson's disease.

Finally, a problem underlying the present invention was the provision ofa method for the assessment of the effects of an agent in the treatmentand/or prevention of a disease, whereby the disease is preferably alysosomal storage disorder and more preferably Gaucher disease.

Similarly, a problem underlying the present invention was the provisionof a method for the assessment of the effects of an agent in thetreatment and/or prevention of a disease, whereby the disease isParkinson's disease.

These and other problems underlying the present invention are solved bythe subject matter of the attached claims. Additionally, these and otherproblems underlying the present invention are solved by the subjectmatter of the following embodiments.

Embodiment 1: Use of lyso-Gb1 as a target in the treatment of a disease.

Embodiment 2: Use of lyso-Gb1 according to embodiment 1, wherein thedisease is a lysosomal storage disorder (LSD).

Embodiment 3: Use according to any one of embodiments 1 to 2, whereinthe lysosomal storage disorder is a lysosomal storage disorder caused bya defective activity of glucocerebrosidase.

Embodiment 4: Use according to any one of embodiments 1 to 3, whereinthe disease is Gaucher disease.

Embodiment 5: Use according to embodiment 4, wherein Gaucher disease isa mild form of Gaucher disease.

Embodiment 6: Use according to embodiment 4, wherein Gaucher disease isnon-neuronopathic Gaucher disease.

Embodiment 7: Use according to embodiment 4, wherein Gaucher disease isa neuronopathic Gaucher disease.

Embodiment 8: Use according to any one of embodiments 1 to 4, whereinthe disease is selected from the group comprising type 1 Gaucherdisease, type 2 Gaucher disease and type 3 Gaucher disease.

Embodiment 9: Use according to embodiment 8, wherein the disease is type1 Gaucher disease.

Embodiment 10: Use according to any one of embodiments 1 to 8, whereinthe disease is type 2 Gaucher disease or type 3 Gaucher disease.

Embodiment 11: Use according to embodiment 1, wherein the disease isParkinson's disease.

Embodiment 12: Use according to any one of embodiments 1 to 11, whereinthe target is a druggable target.

Embodiment 13: Use according to any one of embodiments 1 to 12, whereinthe target is an in vitro target.

Embodiment 14: Use according to any one of embodiments 1 to 13, whereinthe use is an in vitro use.

Embodiment 15: Use according to any one of embodiments 1 to 12, whereinthe target is an in vivo target.

Embodiment 16: Use according to any one of embodiments 1 to 12 and 15,wherein the use is an in vivo use.

Embodiment 17: Use of lyso-Gb1 as a target in the development of a drug,preferably the drug is capable of and/or suitable for treating and/orpreventing a disease

Embodiment 18: Use of lyso-Gb1 according to embodiment 17, wherein thedisease is a lysosomal storage disorder (LSD).

Embodiment 19: Use according to any one of embodiments 17 to 18, whereinthe lysosomal storage disorder is a lysosomal storage disorder caused bya defective activity of glucocerebrosidase.

Embodiment 20: Use according to any one of embodiments 17 to 19, whereinthe disease is Gaucher disease.

Embodiment 21: Use according to embodiment 20, wherein Gaucher diseaseis a mild form of Gaucher disease.

Embodiment 22: Use according to any one of embodiments 20 to 21, whereinGaucher disease is non-neuronopathic Gaucher disease.

Embodiment 23: Use according to embodiment 20, wherein Gaucher diseaseis a neuronopathic Gaucher disease.

Embodiment 24: Use according to any one of embodiments 17 to 21, whereinthe disease is selected from the group comprising type 1 Gaucherdisease, type 2 Gaucher disease and type 3 Gaucher disease.

Embodiment 25: Use according to embodiment 24, wherein the disease istype 1 Gaucher disease.

Embodiment 26: Use according to any one of embodiments 17 to 24, whereinthe disease is type 2 Gaucher disease or type 3 Gaucher disease.

Embodiment 27: Use according to embodiment 17, wherein the disease isParkinson's disease.

Embodiment 28: Use according to any one of embodiments 17 to 27, whereinthe target is a druggable target.

Embodiment 29: Use according to any one of embodiments 17 to 28, whereinthe drug is an antagonist of lyso-Gb1.

Embodiment 30: Use according to embodiment 29, wherein the antagonist iscapable of reducing or ameliorating at least one symptom of a disease.

Embodiment 31: Use according to embodiment 29, wherein the antagonist iscapable of treating a disease, preferably in humans.

Embodiment 32: Use according to embodiment 29, wherein the antagonist iscapable of reducing or ameliorating at least one symptom of a disease inan animal model of the disease.

Embodiment 33: Use according to embodiment 29, wherein antagonist iscapable of treating a disease in an animal model of the disease.

Embodiment 34: Use according to any one of embodiments 29 to 33, whereinthe disease is any disease as defined in any one of embodiments 18 to27.

Embodiment 35: Use according to any one of embodiments 32 to 34, whereinthe animal model is a lyso-Gb1 induced animal model.

Embodiment 36: Use according to any one of embodiments 32 to 35, whereinthe animal model is an animal model for the disease.

Embodiment 37: Use according to embodiment 36, wherein the animal modelfor or of the disease is an animal model for a disease as defined in anyone of embodiments 18 to 27.

Embodiment 38: Use according to any one of embodiments 32 to 37, whereinthe animal model is a mammal.

Embodiment 39: Use according to embodiment 38, wherein the mammal is arodent.

Embodiment 40: Use according to embodiment 39, wherein the rodent isselected from the group comprising mouse and rat.

Embodiment 41: Use according to embodiment 38, wherein the mammal isselected from the group comprising a primate, a dog, a pig and a sheep.

Embodiment 42: Use according to any one of embodiments 29 to 41, whereinthe antagonist is capable of reducing at least one peripheral symptom ofthe disease.

Embodiment 43: Use according to embodiment 42, wherein the at least oneperipheral symptom of the disease is selected from the group comprisingvisceral enlargement of the spleen, mild anemia and inflammatory tissueresponse, preferably at least one peripheral symptom of type 1, of type2 and/or of type 3 Gaucher disease, more preferably of type 1 Gaucherdisease.

Embodiment 44: Use according to embodiment 43, wherein the animal modelis a mouse animal model.

Embodiment 45: Use according to embodiment 44, wherein the mouse animalmodel is a lyso-Gb1 induced animal mouse model.

Embodiment 46: Use according to embodiment 45, wherein the lyso-Gb1induced animal mouse model is a lyso-Gb1 induced model for Gaucherdisease, preferably the mouse is selected from the group comprising aC57BL/6JRj mouse, a C57BL/6 mouse and a C57/BL/10 mouse.

Embodiment 47: Use according to embodiment 46, wherein Gaucher diseaseis selected from the group comprising mild form of Gaucher disease, type1 Gaucher disease and non-neuronopathic Gaucher disease.

Embodiment 48: Use according to any one of embodiments 17 to 47, whereinthe drug is selected from the group comprising a small molecule, alyso-Gb 1 binding protein, a lyso-Gb1 binding peptide, an antibody orantigen-binding fragment thereof, an anticalin, an aptamer, aspiegelmer, and a lyso-Gb1 degrading enzyme.

Embodiment 49: Use according to embodiment 48, wherein the antibody isan anti-lyso-Gb1 antibody and the antibody fragment is a lyso-Gb1binding fragment of the antibody.

Embodiment 50: Use according to embodiment 48, wherein the aptamer, thespiegelmer and the anticalin is each binding to or capable of binding tolyso-Gb1.

Embodiment 51: Use of lyso-Gb1 in the generation of an animal model of adisease.

Embodiment 52: Use of lyso-Gb1 according to embodiment 51, wherein thedisease is a lysosomal storage disorder (LSD).

Embodiment 53: Use according to any one of embodiments 51 to 52, whereinthe lysosomal storage disorder is a lysosomal storage disorder caused bya defective activity of glucocerebrosidase.

Embodiment 54: Use according to any one of embodiments 51 to 53, whereinthe disease is Gaucher disease.

Embodiment 55: Use according to embodiment 54, wherein Gaucher diseaseis a mild form of Gaucher disease.

Embodiment 56: Use according to embodiment 55, wherein Gaucher diseaseis non-neuronopathic Gaucher disease.

Embodiment 57: Use according to embodiment 54, wherein Gaucher diseaseis a neuronopathic Gaucher disease.

Embodiment 58: Use according to any one of embodiments 51 to 55, whereinthe disease is selected from the group comprising type 1 Gaucherdisease, type 2 Gaucher disease and type 3 Gaucher disease.

Embodiment 59: Use according to embodiment 58, wherein the disease istype 1 Gaucher disease.

Embodiment 60: Use according to any one of embodiments 51 to 58, whereinthe disease is type 2 Gaucher disease or type 3 Gaucher disease.

Embodiment 61: Use according to any one of embodiments 51 to 58, whereinthe disease is type 3 Gaucher disease.

Embodiment 62: Use according to embodiment 51, wherein the disease isParkinson's disease.

Embodiment 63: Use according to any one of embodiments 51 to 62, whereinthe animal model is a lyso-Gb1 induced animal model.

Embodiment 64: Use according to any one of embodiments 51 to 63, whereinthe animal model is a mammal.

Embodiment 65: Use according to embodiment 64, wherein the mammal is arodent.

Embodiment 66: Use according to embodiment 65, wherein the rodent isselected from the group comprising mouse and rat.

Embodiment 67: Use according to embodiment 64, wherein the mammal isselected from the group comprising a primate, a dog, a pig and a sheep.

Embodiment 68: Use according to embodiment 66, wherein the animal modelis a mouse animal model.

Embodiment 69: Use according to embodiment 68, wherein the mouse animalmodel is a lyso-Gb1 induced mouse animal model.

Embodiment 70: Use according to embodiment 69, wherein the lyso-Gb1induced mouse model is a lyso-Gb1 induced mouse model for Gaucherdisease, preferably the mouse is selected from the group comprising aC57BL/6JRj mouse, a C57BL/6 mouse and a C57/BL/10 mouse.

Embodiment 71: Use according to embodiment 70, wherein Gaucher diseaseis selected from the group comprising mild form of Gaucher disease, type1 Gaucher disease and non-neuronopathic Gaucher disease.

Embodiment 72: An antagonist of lyso-Gb1 for use in the treatment and/orprevention of a disease.

Embodiment 73: The antagonist for use according to embodiment 72,wherein the antagonist is selected from the group comprising a smallmolecule, a lyso-Gb 1 binding protein, a lyso-Gb1 binding peptide, anantibody or antigen-binding fragment thereof, an anticalin, an aptamer,a spiegelmer, and a lyso-Gb1 degrading enzyme.

Embodiment 74: The antagonist for use according to embodiment 73,wherein the antibody is an anti-lyso-Gb1 antibody and the antibodyfragment is a lyso-Gb1 binding fragment of the antibody.

Embodiment 75: The antagonist for use according to embodiment 73,wherein the aptamer and the spiegelmer is each binding to or capable ofbinding to lyso-Gb1.

Embodiment 76: The antagonist for use according to any one ofembodiments 72 to 75, wherein the disease is a lysosomal storagedisorder (LSD).

Embodiment 77: The antagonist for use according to embodiment 76,wherein the lysosomal storage disorder is a lysosomal storage disordercaused by a defective activity of glucocerebrosidase.

Embodiment 78: The antagonist for use according to any one ofembodiments 72 to 77, wherein the disease is Gaucher disease, preferablythe antagonist is capable of reducing at least one peripheral symptom ofthe Gaucher disease, wherein more preferably the at least one peripheralsymptom of the disease is selected from the group comprising visceralenlargement of the spleen, mild anemia and inflammatory tissue response,preferably at least one peripheral symptom of type 1, of type 2 and/orof type 3 Gaucher disease, more preferably of type 1 Gaucher disease.

Embodiment 79: The antagonist for use according to embodiment 78,wherein Gaucher disease is a mild form of Gaucher disease.

Embodiment 80: The antagonist for use according to embodiment 78,wherein Gaucher disease is non-neuronopathic Gaucher disease.

Embodiment 81: The antagonist for use according to embodiment 78,wherein Gaucher disease is a neuronopathic Gaucher disease.

Embodiment 82: The antagonist for use according to any one ofembodiments 72 to 78, wherein the disease is selected from the groupcomprising type 1 Gaucher disease, type 2 Gaucher disease and type 3Gaucher disease.

Embodiment 83: The antagonist for use according to embodiment 82,wherein the disease is type 1 Gaucher disease.

Embodiment 84: The antagonist for use according to any one ofembodiments 72 to 82, wherein the disease is type 2 Gaucher disease ortype 3 Gaucher disease.

Embodiment 85: The antagonist for use according to embodiment 72,wherein the disease is Parkinson's disease.

Embodiment 86: Use of an antagonist of lyso-Gb1 in the manufacture of amedicament.

Embodiment 87: Use according to embodiment 86, wherein the antagonist isselected from the group comprising a small molecule, a lyso-Gb 1 bindingprotein, a lyso-Gb1 binding peptide, an antibody or antigen-bindingfragment thereof, an anticalin, an aptamer, a spiegelmer, and a lyso-Gb1degrading enzyme.

Embodiment 88: Use according to embodiment 87, wherein the antibody isan anti-lyso-Gb1 antibody and the antibody fragment is a lyso-Gb1binding fragment of the antibody.

Embodiment 89: Use according to embodiment 87, wherein the aptamer, thespiegelmer and the anticalin is each binding to or capable of binding tolyso-Gb1.

Embodiment 90: Use according to any one of embodiments 86 to 89, whereinthe disease is a lysosomal storage disorder (LSD).

Embodiment 91: Use according to embodiment 90, wherein the lysosomalstorage disorder is a lysosomal storage disorder caused by a defectiveactivity of glucocerebrosidase.

Embodiment 92: Use according to any one of embodiments 86 to 91, whereinthe disease is Gaucher disease, preferably the medicament is capable ofreducing at least one peripheral symptom of Gaucher disease, whereinmore preferably the at least one peripheral symptom of the disease isselected from the group comprising visceral enlargement of the spleen,mild anemia and inflammatory tissue response, more preferably at leastone peripheral symptom of type 1 Gaucher disease, of type 2 and/or oftype 3 Gaucher disease, most preferably of type 1 Gaucher disease.

Embodiment 93: Use according to embodiment 91, wherein Gaucher diseaseis a mild form of Gaucher disease.

Embodiment 94: Use according to embodiment 92, wherein Gaucher diseaseis non-neuronopathic Gaucher disease.

Embodiment 95: Use according to embodiment 92, wherein Gaucher diseaseis a neuronopathic Gaucher disease.

Embodiment 96: Use according to any one of embodiments 86 to 92, whereinthe disease is selected from the group comprising type 1 Gaucherdisease, type 2 Gaucher disease and type 3 Gaucher disease.

Embodiment 97: Use according to embodiment 96, wherein the disease istype 1 Gaucher disease.

Embodiment 98: Use according to any one of embodiments 86 to 96, whereinthe disease is type 2 Gaucher disease or type 3 Gaucher disease.

Embodiment 99: Use according to embodiment 86, wherein the disease isParkinson's disease.

Embodiment 100: A method for the generation of an animal model for adisease, wherein the method comprises

a) administering to an animal lyso-Gb1 over a period of time,

b) determining whether the animal shows at least one symptom of thedisease.

Embodiment 101: The method according to embodiment 100, wherein thedisease is a lysosomal storage disorder (LSD).

Embodiment 102: The method according to embodiment 101, wherein thelysosomal storage disorder is a lysosomal storage disorder caused by adefective activity of glucocerebrosidase.

Embodiment 103: The method according to any one of embodiments 100 to102, wherein the disease is Gaucher disease.

Embodiment 104: The method according to embodiment 102, wherein Gaucherdisease is a mild form of Gaucher disease.

Embodiment 105: The method according to embodiment 103, wherein Gaucherdisease is non-neuronopathic Gaucher disease.

Embodiment 106: The method according to embodiment 103, wherein Gaucherdisease is a neuronopathic Gaucher disease.

Embodiment 107: The method according to any one of embodiments 100 to103, wherein the disease is selected from the group comprising type 1Gaucher disease, type 2 Gaucher disease and type 3 Gaucher disease.

Embodiment 108: The method for use according to embodiment 107, whereinthe disease is type 1 Gaucher disease.

Embodiment 109: The method according to any one of embodiments 100 to107, wherein the disease is type 2 Gaucher disease or type 3 Gaucherdisease.

Embodiment 110: The method according to embodiment 100, wherein thedisease is Parkinson's disease.

Embodiment 111: The method according to any one of embodiments 100 to110, wherein the animal model is a lyso-Gb1 induced animal model.

Embodiment 112: The method according to any one of embodiments 100 to111, wherein the animal model is a mammal.

Embodiment 113: The method according to embodiment 112, wherein themammal is a rodent.

Embodiment 114: The method according to embodiment 113, wherein therodent is selected from the group comprising mouse and rat.

Embodiment 115: The method according to embodiment 113, wherein themammal is selected from the group comprising a primate, a dog, a pig anda sheep.

Embodiment 116: The method according to embodiment 112, wherein theanimal model is a mouse animal model.

Embodiment 117: The method according to embodiment 116, wherein themouse animal model is a lyso-Gb1 induced mouse animal model.

Embodiment 118: The method according to embodiment 117, wherein thelyso-Gb1 induced mouse model is a lyso-Gb1 induced animal mouse modelfor Gaucher disease.

Embodiment 119: The method according to embodiment 118, wherein Gaucherdisease is selected from the group comprising mild form of Gaucherdisease, type 1 Gaucher disease and non-neuronopathic Gaucher disease.

Embodiment 120: The method according to any one of embodiments 100 to119, wherein lyso-Gb1 is administered until the animal shows at leastone symptom of the disease.

Embodiment 121: The method according to any one of embodiments 100 to120, wherein lyso-Gb1 is administered subcutaneously.

Embodiment 122: The method of any one of embodiments 100 to 121,preferably embodiment 121, wherein lyso-Gb1 is administered over aperiod of time of at least two weeks, at least four weeks, at least sixweeks, at least eight weeks, at least 10 weeks, or at least twelveweeks.

Embodiment 123: The method of any one of embodiments 100 to 121,preferably any one of embodiments 121 to 122, wherein lyso-Gb1 isadministered over a period of time of two weeks, four weeks, six weeks,eight weeks, 10 weeks or twelve weeks.

Embodiment 124: The method of any one of embodiments 100 to 123,preferably any one of embodiments 121 to 123, the animal is a mouse.

Embodiment 125: The method of embodiment 124, wherein the mouse isselected from the group comprising a C57BL/6JRj mouse, a C57BL/6 mouseand a C57/BL/10 mouse.

Embodiment 126: The method of any one of embodiments 100 to 125,preferably any one of embodiments 121 to 125, wherein the at least onesymptom is a peripheral symptom.

Embodiment 127: The method of any one of embodiments 100 to 126,preferably embodiment 126, wherein the peripheral symptom is selectedfrom the group comprising visceral enlargement of the spleen, mildanemia, inflammatory tissue response and visceral enlargement of theliver.

Embodiment 128: The method of any one of embodiments 100 to 127,preferably any one of embodiments 121 to 127, wherein lyso-Gb1 isadministered until the lyso-Gb1 concentration in the peripheral blood ofthe animal is about 500 ng/ml peripheral blood or more.

Embodiment 129: The method of any one of embodiments 100 to 128,preferably any one of embodiments 121 to 128, wherein the disease isGaucher disease selected from the group comprising mild form of Gaucherdisease, type 1 Gaucher disease and non-neuronopathic Gaucher disease.

Embodiment 130: The method according to any one of embodiments 100 to120, wherein lyso-Gb1 is administered intrathecally.

Embodiment 131: The method of any one of embodiments 100 to 120 and 130,preferably embodiment 130, wherein lyso-Gb1 is administered over aperiod of time of at least two weeks, at least four weeks, at least sixweeks, at least eight weeks, at least 10 weeks, or at least twelveweeks.

Embodiment 132: The method of any one of embodiments 100 to 120 and 130to 131, preferably any one of embodiments 130 to 131, wherein lyso-Gb1is administered over a period of time of two weeks, four weeks, sixweeks, eight weeks, 10 weeks or twelve weeks.

Embodiment 133: The method of any one of embodiments 100 to 120 and 130to 132, preferably any one of embodiments 130 to 1323, the animal is amouse.

Embodiment 134: The method of embodiment 133, wherein the mouse is aC57BL/6JRj mouse, a C57BL/6 mouse and a C57/BL/10 mouse Embodiment 135:The method of any one of embodiments 100 to 120 and 130 to 134,preferably any one of embodiments 130 to 134, wherein the at least onesymptom is a central nervous symptom.

Embodiment 136: The method of any one of embodiments 100 to 120 and 130to 135, preferably embodiment 135, wherein the central symptom isselected from the group comprising ataxia, dementia, ocular apraxia, andparkinsonism.

Embodiment 137: The method of any one of embodiments 100 to 120 and 130to 136, preferably any one of embodiments 130 to 136, wherein lyso-Gb1is administered until the lyso-Gb1 concentration in the peripheral bloodof the animal is about 10 to 30 ng/ml cerebrospinal fluid.

Embodiment 138: The method of any one of embodiments 100 to 120 and 130to 137, preferably any one of embodiments 130 to 137, wherein thedisease is Gaucher disease selected from the group comprising type 2Gaucher disease, type 3 Gaucher disease and neuronopathic Gaucherdisease.

Embodiment 139: An animal model obtainable by a method according to anyone of embodiments 100 to 138, wherein the animal model is an animalmodel for a disease, preferably the disease is a lysosomal storagedisease.

Embodiment 140: An animal model, wherein the animal model is a lyso-Gb1induced animal model for a disease, preferably the disease is alysosomal storage disease.

Embodiment 141: The animal model according to any one of embodiments 139to 140, wherein the lysosomal storage disorder is a lysosomal storagedisorder caused by a defective activity of glucocerebrosidase.

Embodiment 142: The animal model according to any one of embodiments 139to 141, wherein the disease is Gaucher disease, preferably the diseaseis at least one peripheral symptom of Gaucher disease, whereinpreferably the at least one peripheral symptom of Gaucher disease isselected from the group comprising visceral enlargement of the spleen,mild anemia and inflammatory tissue response, more preferably at leastone peripheral symptom of type 1, of type 2 and/or of type 3 Gaucherdisease, most preferably of type 1 Gaucher disease.

Embodiment 143: The animal model according to embodiment 142, whereinGaucher disease is a mild form of Gaucher disease.

Embodiment 144: The animal model according to embodiment 142, whereinGaucher disease is non-neuronopathic Gaucher disease.

Embodiment 145: The animal model according to embodiment 142, whereinGaucher disease is a neuronopathic Gaucher disease.

Embodiment 146: The animal model according to any one of embodiments 139to 142, wherein the disease is selected from the group comprising type 1Gaucher disease, type 2 Gaucher disease and type 3 Gaucher disease.

Embodiment 147: The animal model according to embodiment 146, whereinthe disease is type 1 Gaucher disease.

Embodiment 148: The animal model according to any one of embodiments 139to 146, wherein the disease is type 2 Gaucher disease or type 3 Gaucherdisease.

Embodiment 149: The animal model according to any one of embodiments 139and 140, wherein the disease is Parkinson's disease.

Embodiment 150: The animal model according to any one of embodiments 139to 149, wherein the animal model is a lyso-Gb1 induced animal model.

Embodiment 151: The animal model according to any one of embodiments 139to 150, wherein the animal model is a mammal.

Embodiment 152: The animal model according to embodiment 151, whereinthe mammal is a rodent.

Embodiment 153: The animal model according to embodiment 151, whereinthe rodent is selected from the group comprising mouse and rat.

Embodiment 154: The animal model according to embodiment 151, whereinthe mammal is selected from the group comprising a primate, a dog, a pigand a sheep.

Embodiment 155: The animal model according to embodiment 151, whereinthe animal model is a mouse animal model.

Embodiment 156: The animal model according to embodiment 155, whereinthe mouse animal model is a lyso-Gb1 induced mouse animal model.

Embodiment 157: The animal model according to embodiment 156, whereinthe lyso-Gb1 induced mouse animal model is a lyso-Gb1 induced mouseanimal model for Gaucher disease.

Embodiment 158: The animal model according to embodiment 157, whereinGaucher disease is selected from the group comprising mild form ofGaucher disease, type 1 Gaucher disease and non-neuronopathic Gaucherdisease.

Embodiment 159: A method for the screening of an agent suitable forand/or capable of treating and/or preventing a disease, wherein themethod comprises

-   -   testing whether a candidate compound is an antagonist of        lyso-Gb1,

wherein if the candidate compound is an antagonist of lyso-Gb1, thecandidate compound is an agent suitable for and/or capable of treatingand/or preventing a disease.

Embodiment 160: The method according to embodiment 159, wherein theantagonist of lyso-Gb1 is an antagonist as defined in any one ofembodiments 72 to 85.

Embodiment 161: The method according to any one of embodiments 159 to160, wherein the candidate compound is selected from a small molecule, alyso-Gb 1 binding protein, a lyso-Gb1 binding peptide, an antibody orantigen-binding fragment thereof, an anticalin, an aptamer, aspiegelmer, a and a lyso-Gb1 degrading enzyme.

Embodiment 162: The method according to any one of embodiments 159 to161, wherein the testing whether a candidate compound is an antagonistfor lyso-Gb1 is made in an animal model according to any one ofembodiments 139 to 158 or is made using an animal model according to anyone of embodiments 139 to 158.

Embodiment 163: The method according to embodiment 162, wherein themethod comprises administering the candidate to the animal model anddetermining whether the candidate compound ameliorates at least onesymptom of the disease, preferably the at least one symptom is at leastone peripheral symptom of Gaucher disease, wherein even more preferablythe at least one peripheral symptom of Gaucher disease is selected fromthe group comprising visceral enlargement of the spleen, mild anemia andinflammatory tissue response, most preferably at least one peripheralsymptom of type 1, of type 2 and/or of type 3 Gaucher disease, utmostpreferably of type 1 Gaucher disease.

Embodiment 164: The method according to any one of embodiments 159 to163, wherein the candidate compound is contained in a library ofcompounds or is taken from a library of compounds.

Embodiment 165: A method for the assessment of the effects of an agentin the treatment and/or prevention of a disease, wherein the methodcomprises

-   -   testing the effect of the agent in an animal model according to        any one of embodiments 139 to 158 or using an animal model        according to any one of embodiments 139 to 158.

Embodiment 166: The method according to embodiment 165, wherein theagent is an antagonist as defined in any one of embodiments 72 to 85.

Embodiment 167: The method according to any one of embodiments 165 to166, wherein the agent is selected from a small molecule, a lyso-Gb 1binding protein, a lyso-Gb1 binding peptide, an antibody orantigen-binding fragment thereof, an anticalin, an aptamer, aspiegelmer, and a lyso-Gb1 degrading enzyme.

Embodiment 168: The method according to any one of embodiments 165 to167, wherein the method comprises administering the agent to the animalmodel and determining whether the agent ameliorates at least one symptomof the disease, preferably the at least one symptom is at least oneperipheral symptom of Gaucher disease, wherein even more preferably theat least one peripheral symptom of Gaucher disease is selected from thegroup comprising visceral enlargement of the spleen, mild anemia andinflammatory tissue response, most preferably at least one peripheralsymptom of type 1, of type 2 and/or of type 3 Gaucher disease, utmostpreferably of type 1 Gaucher disease.

Embodiment 169: The method according to any one of embodiments 159 to168, wherein the disease is a lysosomal storage disorder (LSD).

Embodiment 170: The method according to any one of embodiments 159 to169, wherein the lysosomal storage disorder is a lysosomal storagedisorder caused by a defective activity of glucocerebrosidase.

Embodiment 171: The method according to any one of embodiments 159 to170, wherein the disease is Gaucher disease.

Embodiment 172: The method according to embodiment 171, wherein Gaucherdisease is a mild form of Gaucher disease.

Embodiment 173: The method according to embodiment 171, wherein Gaucherdisease is non-neuronopathic Gaucher disease.

Embodiment 174: The method according to embodiment 171, wherein Gaucherdisease is a neuronopathic Gaucher disease.

Embodiment 175: The method according to any one of embodiments 159 to171, wherein the disease is selected from the group comprising type 1Gaucher disease, type 2 Gaucher disease and type 3 Gaucher disease.

Embodiment 176: The method according to embodiment 175, wherein thedisease is type 1 Gaucher disease.

Embodiment 177: The method according to any one of embodiments 159 to171, wherein the disease is type 2 Gaucher disease or type 3 Gaucherdisease.

Embodiment 178: The method according to any one of embodiment 139 to168, wherein the disease is Parkinson's disease.

The present invention is based on the surprising finding that lyso-Gb1is a target, and more specifically a druggable target, for a lysosomalstorage disorder, preferably for Gaucher disease, and Parkinson disease.Another surprising finding underlying the present invention is that ananimal model can be generated by using lyso-Gb1, more specifically byadministering lyso-Gb1 to an animal over a period of time, whereuponsaid animal will develop at least one of the symptoms characteristic forthe disease with the disease preferably being a lysosomal storagedisorder, preferably Gaucher disease, or Parkinson's disease.

The present invention is further based on the surprising finding thatlyso-Gb1 causes at least one peripheral symptom of Gaucher disease,whereby the at least one peripheral symptom of Gaucher disease isselected from the group comprising visceral enlargement of the spleen,mild anemia and inflammatory tissue response, more preferably at leastone peripheral symptom of type 1, of type 2 and/or of type 3 Gaucherdisease, utmost preferably of type 1 Gaucher disease. This finding issurprising as many other pathophysiological roles have been attributedto sphingolipids (Rotstein et al., 2010, J Lipid Res 51:1247-1262),lyso-sphingolipids (Ballabio et al., 2009, Biochem Biophys Act1793:684-96), and in particular, lyso-Gb1 (Schueler et al., 2003Neurobiol Dis 14: 595-601).

Lyso-Gb1 is also referred to herein as glucosylsphingosine orlyso-glucocerebroside and has the formula (I):

As preferable used herein in connection with each and any embodiment ofeach and any aspect of the present invention, the terms target andtarget molecule are used herein in an interchangeable manner.

As preferably used herein in connection with each and any embodiment ofeach and any aspect of the present invention a target molecule is amolecule which is targeted by another compound, preferably by anantagonist of the target, whereby preferably the compound interactsdirectly with the target molecule. In connection therewith, a directinteraction is preferably a physical interaction between the compoundand the target molecule preferably including an interaction betweenatoms, ions and/or chemical groups or moieties of the target moleculewith atoms, ions and/or chemical groups or moieties of the compound.

As preferably used herein in connection with each and any embodiment ofeach and any aspect of the present invention, a druggable target is atarget molecule which when directly interacting with a compound in asubject suffering from a disease or being at risk of developing suchdisease results in treatment of the disease and/or prevention of thedisease; preferably such compound is an antagonist of the targetmolecule and/or such direct interaction is preferably a physicalinteraction between the compound and the target molecule preferablyincluding an interaction between atoms, ions and/or chemical groups ormoieties of the target molecule with atoms, ions and/or chemical groupsor moieties of the compound.

As preferably used herein in connection with each and any embodiment ofeach and any aspect of the present invention a candidate compoundlibrary is a library of candidate compounds. Preferably, the librarycomprises at least two or more elements, i.e. at least two or morecompounds. Preferably, a candidate compound is a compound whichinteracts directly with a target molecule. In connection therewith, adirect interaction is preferably a physical interaction between thecandidate compound and the target molecule preferably including aninteraction between atoms, ions and/or chemical groups or moieties ofthe target molecule with atoms, ions and/or chemical groups or moietiesof the candidate compound.

As preferably used herein in connection with each and any embodiment ofeach and any aspect of the present invention, a lysosomal storagedisease (LSD) is a rare inherited metabolic disorder that results fromdefects in lysosomal function. LSDs result when a specific organelle inthe body's cells—the lysosome—malfunctions.

As preferably used herein in connection with each and any embodiment ofeach and any aspect of the present invention, glucocerebrosidase is aglucocerebrosidase the activity of which is decreased compared to ahealthy subject because of hereditary deficiency of said enzyme. Saiddeficiency results from recessive mutation(s) in the gene coding forglucocerebrosidase, a specific lysosomal hydrolase (also known asbeta-glucosidase, EC 3.2.1.45, PDB 1OGS) located on chromosome 1 (1q21)and affects both males and females. Different mutations in thebeta-glucosidase determine the remaining activity of the enzyme, and, toa large extent, the phenotype. Glucocerebrosidase is also referred toherein as β-glucocerebrosidase, beta-glucosidase, acid beta-glucosidase,glucosylceramidase or D-glucosyl-N-acylsphingosine glucohydrolase. Theenzyme is a 55.6 KD, 497 amino acids long protein havingglucosylceramidase activity, i.e. the enzyme catalyses the breakdown ofa fatty substance called glucocerebroside by cleavage, i.e. hydrolysis,of a beta-glucosidic linkage of glucocerebroside, which is anintermediate in glycolipid metabolism. Glucocerebroside, also referredto herein as glucosylceramide or Gb1, is a cell membrane constituent ofred and white blood cells. When the enzyme is defective, the substanceaccumulates, particularly in cells of the mononuclear cell lineage. Thisis because macrophages that clear these cells are unable to eliminatethe waste product, which accumulates in fibrils, and turn into so calledGaucher cells, which appear on light microscopy to resemble crumpled-uppaper. Fatty material can accumulate in the spleen, liver, kidneys,lungs, brain and bone marrow.

As preferably used herein in connection with each and any embodiment ofeach and any aspect of the present invention, Gaucher diseaseencompasses three subtypes on the basis of the age of onset and of signsof nervous system involvement. Type 1, the non-neuronopathic form, isthe most common (>90% of patients), and major symptoms includeenlargement of the spleen and liver (hepatosplenomegaly),thrombocytopenia, anaemia and skeletal disease (Mistry and Zimran, Dis.Model. Mech. 2011, November; 4(6): 746-752). Moreover, pronouncedvisceral inflammation in other organs such as the lungs has beenreported, which is associated with the Infiltration of large,characteristic macrophages into the tissues (“Gaucher cells”) andconcomitance of inflammatory cytokines like of TNF-α, IL-1b, IL-6, andChT1. In the neuronopathic forms, i.e. types 2 and 3 which are both alsoreferred to as neuronal GD (nGD), whereby GD stands for Gaucher disease,and which are much more rare than type 1, neurological abnormalities areobserved in addition to visceral symptoms.

In accordance therewith, and as preferably used herein in connectionwith each and any embodiment of each and any aspect of the presentinvention, Gaucher disease encompass Non-neuronopathic type I, alsoreferred to herein as type 1, is the most common form of the disease,occurring in approximately 1 in 50,000 live births. It occurs most oftenamong persons of Ashkenazi Jewish heritage. Symptoms may begin early inlife or in adulthood and include enlarged liver and grossly enlargedspleen (together hepatosplenomegaly); the spleen can rupture and causeadditional complications. Skeletal weakness and bone disease may beextensive. Spleen enlargement and bone marrow replacement cause anemia,thrombocytopenia and leukopenia. The brain is not affectedpathologically, but there may be lung and, rarely, kidney impairment.Diseased subjects in this group usually bruise easily (due to low levelsof platelets) and experience fatigue due to low numbers of red bloodcells. Depending on disease onset and severity, type I patients may livewell into adulthood. Many diseased subjects have a mild form of thedisease or may not show any symptoms.

Chronic neuronopathic type 2, also referred to herein as type 2, canbegin at any time in childhood or even in adulthood, and occurs inapproximately 1 in 100,000 live births. It is characterized by slowlyprogressive but milder neurologic symptoms compared to the acute or type3 version. Major symptoms include an enlarged spleen and/or liver,seizures, poor coordination, skeletal irregularities, eye movementdisorders, blood disorders including anemia and respiratory problems.Patients often live into their early teen years and adulthood.

Acute neuronopathic type 3, also referred to herein as type 3, typicallybegins within 6 months of birth and has an incidence rate ofapproximately 1 in 100,000 live births. Symptoms include an enlargedliver and spleen, extensive and progressive brain damage, eye movementdisorders, spasticity, seizures, limb rigidity, and a poor ability tosuck and swallow. Affected children usually die by age 2.

Preferably, symptoms of Gaucher disease may include enlarged spleen andliver, liver malfunction, skeletal disorders and bone lesions that maybe painful, severe neurologic complications, swelling of lymph nodes and(occasionally) adjacent joints, distended abdomen, a brownish tint tothe skin, anemia, low blood platelets and yellow fatty deposits on thewhite of the eye (sclera). Persons affected most seriously may also bemore susceptible to infection.

As preferably used herein in connection with each and any embodiment ofeach and any aspect of the present invention, a subject is a human.

As preferably used herein in connection with each and any embodiment ofeach and any aspect of the present invention, a mouse animal model and amouse model are used herein in an interchangeable manner.

If, in connection with each and any aspect of the present invention andif not explicitly indicated otherwise, reference is made to a disease ordisorder, such disease and disorder, respectively, is either a lysosomalstorage disorder or Parkinson's disease. It is within the presentinvention that the lysosomal storage disorder is typically a lysosomalstorage disorder caused by a defective activity of glucocerebrosidase.Preferably, defective activity of glucocerebrosidase means that theenzymatic activity of the glucocerebrosidase is reduced compared to theactivity of an enzymatic activity of glucocerebrosidase of a healthyindividual, whereby preferably such individual is a human being. Morepreferably such defective activity of glucocerebrosidase is caused byone or several mutations, preferably amino acid mutations. Insofar,defective activity of glucocerebrosidase means and/or encompassesembodiments where the enzyme is defective compared to the enzyme of ahealthy individual, whereby preferably such individual is a human being.

In an embodiment of each and any aspect of the present invention thelysosomal storage disorder is Gaucher disease. It will be acknowledgedby a person skilled in the art that Gaucher disease exists in variousforms, whereby it is understood in the art that the various formsactually constitute a continuum. Nevertheless, even to date Gaucherdisease is differentiated as or categorized as type 1 Gaucher disease,type 2 Gaucher disease and type 3 Gaucher disease. Type 1 Gaucher ispreferably also referred to as mild form of Gaucher disease ornon-neuronopathic Gaucher disease. In contrast thereto, type 2 Gaucherdisease and type 3 Gaucher disease are also referred to as neuronopathicGaucher disease. It is also within the present invention that Gaucherdisease is a severe form of Gaucher disease.

If, in connection with each and any aspect of the present invention andif not explicitly indicated otherwise, reference is made to an animalmodel, such animal model is preferably an animal model for a disease,preferably for any disease as disclosed herein and more preferably alysosomal storage disease as defined herein including its variousembodiments or Parkinson's disease.

In an embodiment of each and any aspect of the present invention, theanimal model is a mammal. In a preferred embodiment the mammal is arodent, wherein more preferably the rodent is selected from the groupcomprising a mouse, a rat, a rabbit and a Guinea pig. In anotherembodiment the animal is a dog, a sheep, a pig and a primate; preferablythe primate is different from a human being. It will be acknowledged bya person skilled in the art that in accordance with the presentinvention an animal model can be generated based on any animal and anymammal in particular for which lyso-Gb1 is toxic and/or which issusceptible to lysosomal storage diseases and/or which showsglucocerebrosidase activity.

In connection with the present invention the subject undergoing anymethod of treatment and/or any method of prevention in accordance withthe instant invention is preferably a human being. Also, when referenceis made to an organism and without the organism being specified further,such organism is preferably a human being; alternative, such organism isdifferent from a human being.

It will be understood that in a preferred embodiment of any aspect ofthe present invention treatment and/or prevention of Gaucher disease istreatment and/or prevention of at least one peripheral symptom ofGaucher disease, wherein preferably the at least one peripheral symptomof Gaucher disease is selected from the group comprising visceralenlargement of the spleen, mild anemia and inflammatory tissue response,more preferably at least one peripheral symptom of type 1, of type 2and/or of type 3 Gaucher disease, most preferably of type 1 Gaucherdisease.

It will be understood that in a preferred embodiment of any aspect ofthe present invention that a subject to be treated or treatable inaccordance with the present invention is a subject who has beendiagnosed as suffering from Gaucher disease. Methods for the diagnosisare known to the person skilled in the art and, for example, describedin international patent application WO 2012/167925 and in Rolfs A et al.(Rolfs A et al., PLOS ONE, November 2013, vol. 8, issue 11, e79732), thedisclosure of which is incorporated herein by reference. Morespecifically, a subject is diagnosed as suffering from Gaucher diseaseis the concentration of free lyso-Gb1 beyond a cut-off value, whereinthe cut-off value is 5 ng/ml or 12 ng/ml in case the sample is a serumor plasma sample from the subject, whereby such serum or plasma samplemay be one of a dry blood filter card, and wherein the cut-off value is20 ng/ml in case the sample is blood and more preferably whole bloodfrom the subject, whereby such blood or whole blood sample may be one ofa dry blood filter card. As preferably used herein, free lyso-Gb1 refersto lyso-Gb1 which is as such present in a sample from the subject, suchas blood, and, preferably, not the result of a manipulation of thesample of said subject. Such manipulation of a sample can be the onedescribed by Groener et al. (Groener et al. Plasma glucosylceramide andceramide in type 1 Gaucher disease patients: Correlations with diseaseseverity and response to therapeutic intervention. Biochimica etBiophysica Acta 1781(2908)72˜78, 2007). In accordance therewith, freelyso-Gb1 which is present as such in the blood of a subject from whomthe sample is taken, is more particularly not a lyso-Gb1 which isgenerated by chemical, biochemical or physical treatment of the samplecontained in the blood and sample, respectively, preferably outside ofthe body of the patient. It will be also understood by a person skilledin the art that free lyso-Gb1 as used herein, preferably is present inaddition to Gb1 and is a compound produced by the subject's metabolicactivities. Accordingly, Gb1, which is the molecule that is accumulatedin connection with Gaucher's disease is present in the sample from thesubject has compared to the molecule in a free lyso form, i.e.free-lyso-Gb1, present in the blood of the subject at least one fattyacid moiety linked to the primary amino group of the sphingosine moietyof lyso-Gb1.

If, in connection with each and any aspect of the present invention andif not explicitly indicated otherwise, reference is made to anantagonist of lyso Gb1, such antagonist is, in one embodiment, capableof reducing or ameliorating at least one symptom of a disease. I afurther embodiment, the capable of or suitable for treating a disease.In an embodiment, the antagonist is capable of reducing or amelioratingat least one symptom of a disease in an animal model of the disease,preferably the animal model is an animal model of the present invention.In an embodiment, the antagonist is capable of or suitable for treatinga disease in an animal model of the disease, preferably the animal modelis an animal model of the present invention in its various embodiments.In an embodiment of the antagonist of lyso-Gb1 the disease is anydisease as disclosed herein and more preferably a lysosomal storagedisease as defined herein including its various embodiments orParkinson's disease.

In an embodiment, the antagonist is a compound selected from the groupcomprising a small molecule, a lyso-Gb 1 binding protein, a lyso-Gb1binding peptide, an antibody or antigen-binding fragment thereof, anaptamer, a spiegelmer and a lyso-Gb1 degrading enzyme.

In an embodiment, a small molecule is a chemical compound complying withthe Lepinski's rules of five known to the ones skilled in the art. Morespecifically, according to said Lepinski's rules of a chemical compoundqualifying as a small molecule has no more than one violation of thefollowing criteria: (a) No more than 5 hydrogen bond donors (the totalnumber of nitrogen-hydrogen and oxygen-hydrogen bonds); (b) no more than10 hydrogen bond acceptors (all nitrogen or oxygen atoms), (c) amolecular mass less than 500 Daltons; and (d) an octanol-water partitioncoefficient log P not greater than 5.

In an embodiment, a lyso-Gb1 binding protein is a protein which bindslyso-Gb1 to an extent such that at least one of the above criteria interms of reducing or ameliorating at least one symptom of a/the diseaseand, respective, treating a/the disease, each in the diverse embodimentsdisclosed herein, is fulfilled. In an embodiment the protein is apolymer comprising a chain of amino acid covalently linked through apeptide bond, whereby the polymer comprises about 100 amino acidresidues or more. An embodiment of a lyso-Gb1 binding protein is alyso-Gb1 binding antibody or lyso-Gb1 binding fragment thereof. Afurther lyso-Gb1 binding protein is a lyso-Gb1 binding anticalin.

Methods for the generation of an antibody binding to a distinct target,are known in the art and, for example, described in Harlow, E., andLane, D., “Antibodies: A Laboratory Manual,” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988). An antibody may be amonoclonal antibody or a polyclonal antibody. Preferably, monoclonalantibodies may be manufactured according to the protocol of Cesar andMilstein and further developments based thereon. Antibodies as usedherein, include, but are not limited to, complete antibodies, antibodyfragments or derivatives such as Fab fragments, Fc fragments andsingle-stranded antibodies, as long as they are suitable and capable ofbinding to lyso-Gb1. The generation of polyclonal antibodies is alsoknown to the one skilled in the art and, for example, described inHarlow, E., and Lane, D., “Antibodies: A Laboratory Manual,” Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., (1988). Preferably, theantibodies used for therapeutic purposes are humanized or humanantibodies as defined above.

The antibodies of the present invention and which may be used accordingto the present invention may have one or several markers or labels. Suchmarkers or labels may be useful to detect the antibody either in itsdiagnostic application or its therapeutic application. Preferably themarkers and labels are selected from the group comprising avidine,streptavidine, biotin, gold and fluorescein and used, e.g., in ELISAmethods. These and further markers as well as methods are, e.g.described in Harlow, E., and Lane, D., “Antibodies: A LaboratoryManual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(1988).

It is also within the present invention that the label or markerexhibits an additional function apart from detection, such asinteraction with other molecules. Such interaction may be, e.g.,specific interaction with one or several other compounds. These othercompounds may either be those inherent to the system where the antibodyis used such as the human or animal body or the sample which is analysedby using the respective antibody. Appropriate markers may, for example,be biotin or fluorescein with the specific interaction partners thereofsuch as avidine and streptavidine and the like being present on therespective compound or structure to interact with the thus marked orlabelled antibody.

In an embodiment, a lyso-Gb1 binding peptide is a protein which bindslyso-Gb1 to an extent such that at least one of the above criteria interms of reducing or ameliorating at least one symptom of a/the diseaseand, respective, treating a/the disease, each in the diverse embodimentsdisclosed herein, is fulfilled. In an embodiment the peptide is apolymer comprising a chain of amino acid covalently linked through apeptide bond, whereby the polymer comprises less than 100 amino acidresidues. As used herein, the term peptide comprises also polypeptidestypically comprising from about 10 to about 100 amino acid residues, andpeptide in the narrower sense typically comprising 2 to about 10 aminoacid residues. In an embodiment, lyso-Gb1 binding peptide is a lyso-Gb1binding anticalin.

Anticalins a target binding polypeptides which are, among others,described in German patent application DE 197 42 706.

Aptamers are D-nucleic acids which are either single stranded or doublestranded and which specifically interact with a target molecule. Themanufacture or selection of aptamers is, e.g., described in Europeanpatent EP 0 533 838. Basically, the following steps are realized. First,a mixture of nucleic acids, i.e. potential aptamers, is provided wherebyeach nucleic acid typically comprises a segment of several, preferablyat least eight subsequent randomised nucleotides. This mixture issubsequently contacted with the target molecule whereby the nucleicacid(s) bind to the target molecule, such as based on an increasedaffinity towards the target or with a bigger force thereto, compared tothe candidate mixture. The binding nucleic acid(s) are/is subsequentlyseparated from the remainder of the mixture. Optionally, the thusobtained nucleic acid(s) is amplified using, e.g. polymerase chainreaction. These steps may be repeated several times giving at the end amixture having an increased ratio of nucleic acids specifically bindingto the target from which the final binding nucleic acid is thenoptionally selected. These specifically binding nucleic acid(s) arereferred to aptamers. It is obvious that at any stage of the method forthe generation or identification of the aptamers samples of the mixtureof individual nucleic acids may be taken to determine the sequencethereof using standard techniques. It is within the present inventionthat the aptamers may be stabilized such as, e.g., by introducingdefined chemical groups which are known to the one skilled in the art ofgenerating aptamers. Such modification may for example reside in theintroduction of an amino group at the 2′-position of the sugar moiety ofthe nucleotides. Aptamers are currently used as therapeutical agents.However, it is also within the present invention that the thus selectedor generated aptamers may be used for target validation and/or as leadsubstance for the development of medicaments, preferably of medicamentsbased on small molecules. This is actually done by a competition assaywhereby the specific interaction between the target molecule and theaptamer is inhibited by a candidate drug whereby upon replacement of theaptamer from the complex of target and aptamer it may be assumed thatthe respective drug candidate allows a specific inhibition of theinteraction between target and aptamer, and if the interaction isspecific, said candidate drug will, at least in principle, be suitableto block the target and thus decrease its biological availability oractivity in a respective system comprising such target. The thusobtained small molecule may then be subject to further derivatisationand modification to optimise its physical, chemical, biological and/ormedical characteristics such as toxicity, specificity, biodegradabilityand bioavailability. In a preferred embodiment, the aptamer is anaptamer capable of binding to lyso-Gb1.

The generation or manufacture of spiegelmers which may be used orgenerated according to the present invention is based on a similarprinciple. The manufacture of spiegelmers is described in theinternational patent application WO 98/08856. Spiegelmers are L-nucleicacids, which means that they are composed of L-nucleotides rather thanaptamers which are composed of D-nucleotides as aptamers are.Spiegelmers are characterized by the fact that they have a very highstability in biological system and, comparable to aptamers, specificallyinteract with the target molecule against which they are directed. Inthe purpose of generating spiegelmers, a heterogonous population ofD-nucleic acids is created and this population is contacted with theoptical antipode of the target molecule which is lyso-Gb1. Subsequently,those D-nucleic acids are separated which do not interact with theoptical antipode of the target molecule. However, those D-nucleic acidsinteracting with the optical antipode of the target molecule areseparated, optionally determined and/or sequenced and subsequently thecorresponding L-nucleic acids are synthesized based on the nucleic acidsequence information obtained from the D-nucleic acids. These L-nucleicacids which are identical in terms of sequence with the aforementionedD-nucleic acids interacting with the optical antipode of the targetmolecule, will specifically interact with the naturally occurring targetmolecule rather than with the optical antipode thereof. Similar to themethod for the generation of aptamers it is also possible to repeat thevarious steps several times and thus to enrich those nucleic acidsspecifically interacting with the optical antipode of the targetmolecule.

In connection with the various aspects of the present invention it isindicated that the disease is a lysosomal storage disease, preferablyGaucher disease. The instant application provides experimental evidencein terms of lyso-Gb1 being a target, preferably a druggable target, andcapable of inducing and generating, respectively, an animal model forsuch disease. Based on said experimental evidence and the titre oflyso-Gb1 observed in patients suffering from Parkinson's disease, it isacknowledged and appreciated by a person skilled in the art that thesame functions annotated to and exercised by lyso-Gb1 in connection withlysosomal storage disorder and Gaucher disease in particular, will beequally shown and exercised by lyso-Gb1 in Parkinson's disease. In anembodiment, Parkinson's disease is Parkinson's disease of a subjecthaving a mutant glucocerebrosidase, preferably a mutantglucocerebrosidase as observed in subjects suffering from Gaucherdisease, whereby more preferably the subject suffering from Parkinson'sdisease does not show any clinical sign of Gaucher disease.

The lyso-Gb1 degrading enzyme is in an embodiment a lyso-Gb1metabolizing enzyme. Such kind of enzyme is suitable for use as anantagonist of lyso-Gb1 under the proviso that the lyso-Gb1 derivativeobtained upon the enzyme having acted on lyso-Gb1 is no longer anantagonist of lyso-Gb1 is defined in connection with the presentinvention.

In a first aspect, the problem underlying the present invention issolved by the use of lyso-Gb1 as a target in the treatment of a disease.Such use is also referred to herein as the use according to theinvention, the therapeutic use of lyso-Gb1 according to the invention,or the first use according to the present invention. Such first aspectis based on the surprising finding that lyso-Gb1 which has beenidentified recently as a diagnostic marker for Gaucher disease (Dekker Net al., Blood. 118(16):e118-27; Rolfs A et al., PLoS One. 8(11):e79732)is a target in a disease, preferably a lysosomal storage disease in itsvarious embodiments disclosed herein, and Parkinson's disease. Moreimportantly, lyso-Gb1 is also a druggable target. As preferably usedherein, a druggable target is known to or is predicted to bind with highaffinity to a drug.

Furthermore, by definition, the binding of the drug to a druggabletarget must alter the function of the target with a therapeutic benefitto the patient.

The use according to the first aspect of the present inventionencompasses both in vitro and in vivo use. In the embodiment where theuse is an in vitro use, lyso-Gb1 is used in a chemical, biochemical,physical or cell-based assay. Such assay determines, for example,whether a drug candidate is effective in binding to lyso-Gb1 and/orinterfering with its action resulting in one or several of the symptomsof the disease. In an embodiment, the target is present outside of acell, tissue, organ or organism; in an alternative embodiment, thetarget is present within a cell, tissue, organ or organism; such cell,tissue, organ or organism can be either alive or dead. In a furtherembodiment, the use is an in vivo use, i.e. the lyso-Gb1 as target istargeted for the purpose of treating and/or preventing a disease;preferably, in such embodiment lyso-Gb1 being present in an organism,organ, tissue or cell, which are more preferably alive. In analternative embodiment, the in vivo use of lyso-Gb1 as target is not forthe purpose of treatment and/or prevention of a disease; preferably,such in vivo use is for obtaining medical and/or scientific insights asto the involvement of lyso-Gb1 in a cell, tissue, organ or an organism.

In a second aspect, the problem underlying the present invention issolved by the use of lyso-Gb1 as a target in the development of a drug,whereby, preferably, the drug is capable of and/or suitable for treatingand/or preventing a disease preferably a lysosomal storage disease inits various embodiments disclosed herein, and Parkinson's disease. Likethe first aspect, this second aspect is based on the surprising findingthat lyso-Gb1 is not only a diagnostic marker but a target in a disease,preferably a lysosomal storage disease in its various embodimentsdisclosed herein, and Parkinson's disease. Such second aspect is alsoreferred to herein as the use according to the present invention, morespecifically, the second use according to the present invention.

It will be understood that any embodiment of the first aspect of thepresent invention is also an embodiment of the second aspect of thepresent invention and vice versa.

As preferably used herein, the term “development of a drug” preferablymeans any process which provides a drug which is capable of and/orsuitable for treating, preferably, a lysosomal storage disease in itsvarious embodiments disclosed herein, and Parkinson's disease. Suchprocess encompasses any screening for a drug, testing of any drugcandidate as to their usefulness or fitness for a distinct purpose,including quality control, preferably quality control. Such developmentof a drug is different from preparation of a medicament, wherebypreferably preparation of a medicament means formulating and/or packingof a drug so that it is ready for use by a patient or health careworker. In connection with such second aspect of the present invention,the drug, in an embodiment, is an antagonist according to the presentinvention.

In a third aspect, the problem underlying the present invention issolved by the use of lyso-Gb1 in the generation of an animal model of adisease, preferably a lysosomal storage disease in its variousembodiments disclosed herein, and Parkinson's disease. This third aspectis based on the surprising finding that an animal model for this kind ofdisease can be generated by using lyso-Gb1. Such third aspect is alsoreferred to herein as the use according to the present invention, morespecifically, the third use according to the present invention.

The animal used in the generation is any animal disclosed herein. Theanimal model obtained by using lyso-Gb1 in accordance with the thirdaspect of the present invention is also referred to as lyso-Gb1 inducedanimal model. The disease for which the animal model is a model is alysosomal storage disease as disclosed herein, or Parkinson's disease.

In a fourth aspect, the problem underlying the present invention issolved by an antagonist of lyso-Gb1 for use in the treatment and/orprevention of a disease, preferably a lysosomal storage disease in itsvarious embodiments disclosed herein, and Parkinson's disease. Thisfourth aspect is based on the surprising finding that lyso-Gb1 is notonly a diagnostic marker but a druggable target in a disease, preferablya lysosomal storage disease in its various embodiments disclosed herein,and Parkinson's disease. Such fourth aspect is also referred to hereinas the antagonists according to the present invention.

As disclosed herein, the antagonist of the present invention can beprepared using routine measures and without any undue burden in light ofthe above surprising finding underling the present invention. In anembodiment, an antagonist of lyso-Gb1 is an antagonist preventing orameliorating at least one disease related symptom shown by the animalmodel of the present invention.

In a fifth aspect, the problem underlying the present invention issolved by the use of an antagonist of lyso-Gb1 in the manufacture of amedicament, whereby the medicament is for treatment and/or prevention ofa disease, preferably a lysosomal storage disease in its variousembodiments disclosed herein, and Parkinson's disease. Like the fourthaspect of the present invention, this fifth aspect is based on thesurprising finding that lyso-Gb1 is not only a diagnostic marker but adruggable target in a disease, preferably a lysosomal storage disease inits various embodiments disclosed herein, and Parkinson's disease. Suchfifth aspect is also referred to herein as the use according to thepresent invention, more specifically, the fifth use according to thepresent invention.

In a sixth aspect, the problem underlying the present invention issolved by a method for the generation of an animal model for a disease,wherein the method comprises

a) administering to an animal lyso-Gb1 over a period of time,

b) determining whether the animal shows at least one symptom of thedisease,

preferably a lysosomal storage disease in its various embodimentsdisclosed herein, and Parkinson's disease. This sixth aspect is based onthe surprising finding that an animal model for this kind of disease canbe generated by using lyso-Gb1, wherein the animal model is an animalmodel for a disease, preferably a lysosomal storage disease in itsvarious embodiments disclosed herein, and Parkinson's disease. Suchsixth aspect is also referred to herein as the method for the generationof an animal model according to the present invention. As the animalmodel is generated by the use of lyso-Gb1, the animal model obtained orobtainable from such method of the present invention is also referred toas lyso-Gb1 induced animal model.

In an embodiment, lyso-Gb1 is continuously administered over the periodof time.

In an embodiment of the method for the generation of an animal modelaccording to the present invention, the animal provided in step a) istypically a healthy animal which is treated so to develop the phenotypeof the disease, at least one symptom of said disease.

A preferred animal is mouse. It will be acknowledged by a person skilledin the art that typically, a mouse of young age such as 10 weeks isused. It is, however, also within the present invention that an evenyounger or older mouse is used.

In an embodiment according to the present invention, lyso-Gb1 isadministered to the animal until at least one symptom of the disease isdisplayed by the animal. Preferably, the at least one symptom of thedisease is stably displayed. As preferably used herein, stably displayedmeans that the at least one symptom of the disease is displayed by theanimal for a time sufficient so as to carry out the tests and proceduresfor which the animal model is used.

It will be acknowledged by a person skilled in the art that theconcentration of lyso-Gb1 administered to the animal depends on theperiod of time over which lyso-Gb1 is administered and, respectively, tobe administered, and the desired and intended, respectively, severity ofthe disease. In light of the instant disclosure, the concentration maybe determined by routine experiments. Preferably, lyso-Gb1 isadministered to an extent such that an intoxication, preferably achronic intoxication is achieved. In case of non-neuronopathic Gaucherdisease, systemic chronic intoxication is to be achieved, whereas incase of neuronopathic Gaucher disease chronic CNS intoxication is to beachieved.

It will be acknowledged by a person skilled in the art that fornon-neuronopathic Gaucher disease subcutaneous administration ispreferred, whereas for neuronopathic Gaucher disease intrathecaladministration is preferred. In case of subcutaneous administration apossible daily dosage is 10 mg lyso-Gb1/kg body weight, whereby thelyso-Gb1 is continuously administered subcutaneously by means of pumpswith a flow rate of 2.64 μl/day.

As to the specific animal strain and mouse strain used in the method ofthe present invention for the generation of an animal model it will beacknowledged by a person skilled in the art that, in principle, any wildtype laboratory strain can be used. Such wild type laboratory strand isone selected from the group comprising C57BL/6JRj strain, a C57BL/6strain, and C57/BL/10 strain.

In an embodiment, the at least one symptom of the disease, preferably incase of the disease being Gaucher disease and more preferablynon-neuronopathic Gaucher disease, is a peripheral symptom. Preferably,such peripheral symptom is one selected from the group comprisingvisceral enlargement of the spleen, mild anemia, inflammatory tissueresponse and visceral enlargement of the liver. As to enlargement of theliver and spleen, it will be acknowledged by a person skilled in the artthat such enlargement is relative to a non-lyso-Gb1 treated, animalwhich thus effectively acts as a negative control. As preferably usedherein, anemia is defined as “Hb values 2×SD below the baseline value”(Raabe B M et al., J Am Assoc Lab Anim Sci. 50(5):680-5). Mild form ofanemia is this one where the observed Hb values are not much below, i.e.close to the baseline value. As preferably used herein, inflammatorytissue response is determined based on CD68 and F4/80 as known in theart (see, e.g., Boven L A et al., Am J. Clin Pathol 2004 122(3):359-69).

In an embodiment, the at least one symptom of the disease, preferably incase of the disease being Gaucher disease and more preferablyneuronopathic Gaucher disease, is central nervous symptom. Preferably,such central nervous symptom is one selected from the group comprisingataxia, dementia, ocular apraxia, and parkinsonism.

In a seventh aspect, the problem underlying the present invention issolved by an animal model obtainable by a method according to the sixthaspect, wherein the animal model is an animal model for a disease,preferably a lysosomal storage disease in its various embodimentsdisclosed herein, and Parkinson's disease. Like the sixth aspect of thepresent invention, this seventh aspect is based on the surprisingfinding that an animal model for this kind of disease can be generatedby using lyso-Gb1. Such seventh aspect is also referred to herein as theanimal model according to the present invention.

In an eighth aspect, the problem underlying the present invention issolved by an animal model, wherein the animal model is a lyso-Gb1induced animal model for a disease, preferably a lysosomal storagedisease in its various embodiments disclosed herein, and Parkinson'sdisease. Like the seventh aspect of the present invention, this eighthaspect is based on the surprising finding that an animal model for thiskind of disease can be generated by using lyso-Gb1. Such eighth aspectis also referred to herein as the animal model according to the presentinvention.

In a ninth aspect, the problem underlying the present invention issolved by a method for the screening of an agent suitable for and/orcapable of treating and/or preventing a disease, wherein the methodcomprises

-   -   testing whether a candidate compound is an antagonist of        lyso-Gb1,

wherein if the candidate compound is an antagonist of lyso-Gb1, thecandidate compound is an agent suitable for and/or capable of treatingand/or preventing a disease. Preferably, the disease is a lysosomalstorage disease in its various embodiments disclosed herein, andParkinson's disease. This ninth aspect is based on the surprisingfinding that that lyso-Gb1 is not only a diagnostic marker but adruggable target in a disease, preferably a lysosomal storage disease inits various embodiments disclosed herein, and Parkinson's disease. Suchninth aspect is also referred to herein as the method for the screeningof an agent suitable for and/or capable of treating and/or preventing adisease according to the present invention.

In an embodiment of the method for the screening of an agent suitablefor and/or capable of treating and/or preventing a disease according tothe present invention, the testing comprises the use of an animal modelof the present invention. Preferably, said testing comprisesadministering to the animal model the candidate compound and assessingwhether the candidate compound reduces or ameliorates at least onesymptom of the disease displayed by the animal model.

In an embodiment of the method for the screening of an agent suitablefor and/or capable of treating and/or preventing a disease according tothe present invention, the antagonist of lyso-Gb1 is an antagonistaccording to the present invention. In another embodiment, the candidatecompound is a compound small molecule, a lyso-Gb 1 binding protein, alyso-Gb1 binding peptide, an antibody or antigen-binding fragmentthereof, an anticalin, an aptamer, a spiegelmer, a and a lyso-Gb1degrading enzyme.

In a tenth aspect, the problem underlying the present invention issolved by a method for the assessment of the effects of an agent in thetreatment and/or prevention of a disease, Wherein the method comprises

-   -   testing the effect of the agent in an animal model according to        the present invention or using an animal model according to the        present invention.

Preferably, the disease is a lysosomal storage disease in its variousembodiments disclosed herein, and Parkinson's disease. This tenth aspectis based on the surprising finding that lyso-Gb1 is not only adiagnostic marker but a druggable target in a disease, preferably alysosomal storage disease in its various embodiments disclosed herein,and Parkinson's disease. Such tenth aspect is also referred to herein asthe method for the assessment of the effects of an agent in thetreatment and/or prevention of a disease according to the presentinvention.

The method for the assessment of the effects of an agent in thetreatment and/or prevention of a disease according to the presentinvention is, in an embodiment, used for assessing one of the followingeffects of the agent: side effects, synergistic effects, toxic effect,effects on various physiological parameters, effects on biochemicalfactors and parameters. Such effects are known to the one skilled in theart and assays for determining such effects are equally known to aperson skilled in the art. It will also be understood by a personskilled in the art that in such method at least a negative control and,optionally a positive control will be subjected to said method. Based onsuch negative control, any advantageous effect associated with a testedagent can be identified and, optionally, quantified. This method of theinvention is, in an embodiment used in the profiling of an agent anddrug candidate, respectively. Such profiling is part of the preparationof a dossier for submission to health authorities when applying for amarket authorization for an agent to be used in the treatment and/orprevention of a disease.

The present invention is now further illustrated by the followingfigures and examples from which further features, embodiments andadvantages may be taken.

More specifically:

FIGS. 1A and 1 B are diagrams showing the level of lyso-Gb1 in blood ofmice treated with either lyso-Gb1 or vehicle alone, as a function oftime with FIG. 1A presenting the results as a bar diagram and FIG. 1Bpresenting the results as a boxplot; mice received subcutaneous lyso-Gb1for 12 weeks using osmotic mini pumps; lyso-Gb1 values were increased atfirst sampling after 4 weeks, and throughout the treatment phasethereafter; vehicle-treated (or pre-dose) animals had lower, yetdetectable lyso-Gb1 values of 1.2-1.5 ng/mL blood; the number of animalstested is indicated in the figure for each data point the boxes indicatethe median and 25th and 75th percentiles, the whiskers of the graph showthe minimum and maximum values.

FIG. 2A is a diagram showing lyso-Gb1 concentration (ng/ml dry tissue)in various tissues, namely heart, kidneys, liver, spleen and brain;lyso-Gb1 treated animals are shown as black, vehicle-treated animals aswhite boxes indicating the min and max values of three independentanimals whereas each organ sample has been measured in duplicates; thewhite and black lines indicate the mean values; all peripheral organs oftreated mice showed strongly elevated levels of lyso-Gb1 compared tocontrol animals;

FIG. 2 B is a diagram showing lyso-Gb1 concentration (ng/ml urine) inurine that was collected for 24 hr from one group of 4 (untreated) andtwo groups of 3 (treated) animals per cage at 6 weeks after treatmentstart. The obtained lyso-Gb1 levels are indicated above the respectivecolumn. Lyso-Gb1 content in the treated animals refer to a calculatedexcretion rate of 0.6 ng*min-1; lyso-Gb1 treated animals are shown asblack, vehicle-treated animals as white boxes, indicating the minimumand maximum values of three animals, whereas each organ sample has beenmeasured in duplicates. The white and black lines indicate the meanvalues.

FIG. 3 is a panel of two diagrams showing blood changes in lyso-Gb1treated C57/BL/6JRj mice for Hb (hemoglobin; left diagram) and Hct(haemato.crit; right diagram); values were derived from 4 control and 6lyso-Gb1 treated mice at each indicated time point (4 weeks, 8 weeks and12 weeks); the black and white boxes indicate the median, 25th and 75thpercentile, the whiskers of the graph show the largest and smallestvalues; differences between treatment groups at each time point wereanalyzed using two-tailed Mann-Whitney test. Left panel: Hb values were2×SD below the baseline value at each time point, indicating mildanemia. Right panel: Het values significantly differed at 4 and 8 weeksafter treatment initiation.

FIG. 4A is a panel of two diagrams showing the result of organ weightanalysis for spleen and liver after 8 and 12 weeks (g/100 g bodyweight); values were derived from 4 control and 6 lyso-Gb1 treated miceat each indicated time point; the black and white boxes indicate themedian, 25th and 75th percentile, the whiskers of the graph show thelargest and smallest values; differences between treatment groups ateach time point were analyzed using two-tailed Mann-Whitney test; thespleens of treated mice were enlarged versus controls 8 and 12 weeksafter treatment initiation. Liver weight was not statisticallysignificantly different compared with controls.

FIG. 4B is a panel of two diagrams illustrating the result of a Westernblot analysis of inflammatory tissue markers F4/80 and CD68 in spleenand liver, whereas a pronounced increase in spleen levels for F4/80 andCD68 was observed in the lyso-Gb1 treated mice after 8 and A similarincrease was noted in the livers of the same animals.

FIG. 4C is a photograph showing half of a spleen from a control andlyso-Gb1 treated animal, whereas The organ samples illustrate the changein size and a distinctive color.

FIGS. 5 A-B are microphotographs showing H&E-stained spleen sectionsfrom vehicle and lyso-Gb1 treated mice showed similar splenicarchitecture with white and red pulp (FIG. 5A, B).

FIGS. 5 C-J are microphotographs showing paraffin sections from spleenof vehicle and lyso-Gb1 treated mice were immunohistochemically analyzedwith anti-CD68 (FIG. 5C, D and high magnification E, F) and anti-F4/80(FIG. 5G, H and high magnification I, J), whereas section from lyso-Gb1treated mice showed a marked increase of the number of CD68 and F4/80positive cells in comparison to vehicle controls.

FIG. 6 shows tables 1A and 1B that comprises all parameters tested inthe study.

FIG. 7 represents bar diagrams showing GCase levels in spleen and liverlysates after 4, 8 and 12 weeks of after treatment initiation.

FIG. 8 are diagrams showing the level of different types ofglucosylceramides in blood of mice treated with either lyso-Gb1 orvehicle alone, after 4 and 8 weeks.

As described supra, animal models of GD display a broad spectrum ofneurological, hematological and visceral phenotypes based on the natureof the genetic defect. Both genetic and non-genetic (chemically-induced)GD mouse models provide informative insights into the disease pathology,often closely resembling the human phenotype (reviewed in Farfel-Beckeret al., 2011, Dis Mod Mech 4:746-52). However, none of the diseasemodels developed so far permit an isolated analysis of the effectsattributable to sphingolipid storage alone.

As shown in the examples the inventors of the present inventions havedeveloped an animal model that allows an isolated analysis of the effectof lyso-Gb1, independently from potential effects of GCase activity,GCase inactivity, glucosylceramide and/or accumulation ofglucosylceramide.

EXAMPLES Example 1: Materials and Methods

Animal Housing and Treatment

Animal experimental procedures were carried out at Pharmacelsus GmbH,Saarbrucken, Germany. Adult male C57BL/6JRj mice (10 weeks old,purchased from Janvier Labs, France) were housed in atemperature-controlled room (20-24° C.) and maintained in a 12 hlight/12 h dark cycle. Food (Ssniff® R/M-H, 10 mm) and water wereprovided ad libitum.

All experimental procedures were approved by and conducted in accordancewith the regulations of the local Animal Welfare authorities. Thelyso-Gb1 stock solution was prepared in DMSO and propylene glycol at 37°C. with ultrasonic bath treatment for 10 min. Subcutaneousadministration of lyso-Gb1 (Matreya LLC, USA) was achieved by the use ofALZET pumps implanted in the back of their necks and set to a flow rateof 2.64 μl/day in order to obtain a daily dosage of 10 mg lyso-Gb1/kgbody weight. The pumps were loaded with the stock solution and primedprior to implantation according to the manufacturer's instructions.

Blood sampling was carried out using Li-heparine blood obtained from thelateral tail vein. At each sampling time point 2 aliquots of 20 μl weretransferred to dried blood spot filter cards (Centogene AG, Rostock,Germany). After the last sampling time point the mice were sacrificed byinhalation of an overdose of isoflurane and organs were removed, frozenand collected for future examinations. Where applicable, organs wereintersected. Half of the organ was immediately frozen in liquidnitrogen. The other half was formalin-fixed and paraffin-embedded forhistologic analysis (Histalim, Montpellier, France).

Sample Preparation and Lyso-Gb1 Determination from Dried Blood Spots(Abbr. DBS)

3 punches of 3.2 mm in diameter were cut using a DBS puncher (PerkinElmer LAS, Germany) and placed in a 2.2 ml round bottom tube (Eppendorf,Germany). 50 μl extraction solution (DMSO:water, 1:1) and 100 μlinternal standard solution with the standard being dissolved in ethanolwere added on top of the paper punches. Samples were mixed for 30seconds and placed in an incubator (Heidolph, Schwabach, Germany) for 30minutes at 37° C. under agitation at 700 rpm. After incubation, thetubes were sonicated for 1 minute at maximum power and then the liquidwas transferred to a AcroPrep Filter Plate with PTFE membrane (PALL,Germany) placed on a 96 well V-shape bottom plate (VWR, Germany). Thesamples were filtrated by centrifugation for 5 minutes at 3,500 rpm in aHermle Z300 plate centrifuge (Hermle Labortechnik, Germany) to removeany solid particles from the solution.

Preparation of Urine Samples for LC/MRM-MS Analysis

25 μL urine aliquots were added to 100 μL of internal standard and 250μL ethanol. The samples were cooled to 4° C. for 1 h to precipitate theurine proteins, after which they were spun in a benchtop centrifuge for3 minutes at 14,500 rpm. The volume of each sample was quantitativelytransferred to a 96-well plate, and further processed as describedabove.

Organ Sample Preparation for Lyso-Gb1 Determination

After extraction from the animal, the organs were immediatelydeep-frozen in liquid nitrogen. The samples were lyophilized (Alpha 2-4LSC, Christ, Osterode am Harz, GER) and powdered with mortar and pistil.Fractions of 2 to 5 mg powder were established in round bottom tubes. Ontop of the powder, 50 μl per mg powder extraction solution was added andthe sample was incubated for 5 minutes at 37° C. under agitation.Subsequently, the samples were frozen in liquid nitrogen for 30 secondsand sonicated with boost function for 5 minutes. The incubation,freezing and sonication steps were repeated 6 times. At the end themixtures were vortexed and aliquots of 25 μl suspension were used forthe lyso-Gb1 determination. Each sample was supplemented with 100 μlInternal Standard (lyso-Gb2, 200 ng/ml) and 250 μl ethanol. The sampleswere cooled at 4° C. for 1 h to precipitate the membrane proteins andspun in a benchtop centrifuge for 3 minutes at 14.5 krpm. The volume ofeach sample was quantitatively transferred to a 96-well filter plate andfurther processed as described above.

LC/MRM-MS Measurements

LC-MRM-MS analyses of the lyso-Gb1 for both DBS and organ extracts wereperformed using a Waters Acquity UPLC (Waters, UK) coupled with anABSciex 5500 TripleQuad mass spectrometer (ABSciex, Darmstadt, Germany).Chromatographic run was performed on a C8 column with pore size of 3 mM(ACE columns, Germany) using a flow rate of 0.9 ml/min preheated at 60°C. The 10 μl extract were injected on the column and the compounds wereeluted using a linear gradient from 40% A (50 mM formic acid in water)to 100% B (50 mM formic acid in acetone:acetonitrile vol. 1:1). Upstreamfrom UPLC a 3:1 flow splitter was added. The following MRM transitionswere monitored: 624.3→282.2 for the internal standard (with DP of 30V,CE of 38 V and CXP of 10 V) and 462.3→282.2 for lyso-Gb1 (with DP of28V, CE of 30 V and CXP of 10 V). MRM-MS analyses were performed inpositive ion mode using the following parameters: CUR gas 40 psi, ISvoltage 5.5 kV, CAD 8 psi, cone temperature 500° C., GS 1 45 psi, GS2 60psi, EP 10 V. For all batches analyzed a standard curve was measuredusing 7 dilutions of lyso-Gb1 in ethanol (concentrations in ng/ml: 0; 5;10; 50; 100; 200; 1000).

Blood Count

Mice were exsanguinated via the retrobulbar venous plexus underisoflurane anesthesia before being sacrificed by overdose inhalation.200 μL EDTA samples of whole blood were drawn from each animal toanalyze aspartate aminotransferase (AST/GOT), leukocyte count,erythrocyte count, hemoglobin (Hb), hematocrit (PCV=packed cell volume,Hct), mean cell volume (MCV), mean cell Hb (MCH), mean cell Hbconcentration. (MCHC) and platelet count (PLT) (IDEXX Bioresearch,Ludwigsburg, Germany).

Cytokine Analysis

The ProcartaPlex® Multiplex Immunoassay (ebioscience, San Diego, Calif.)was carried out following the instructions of the provider. Differencesbetween the animal groups were obtained using automated MAGPIX© analysisdevice software (Luminex, Austin, Tex.) including quality controlcriteria.

Western Blot

Livers and spleens were homogenized in RIPA buffer containing proteinaseand phosphatase inhibitor (Roche, Mannheim, GER). Lysates werecentrifuged at 15,000 g, 4° C. for 15 min to remove insoluble pellet,and supernatant was collected. Protein concentration was measured usingPierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, Mass.).Typically, 100 μg protein was loaded for electrophoresis on a 4%-15%precast Tris-glycine gradient gel (BioRad, Mannheim, GER). The proteinwas subsequently transferred to a nitrocellulose membrane by semidrytransfer apparatus (BioRad, Munich, GER) for immunodetection analysis.The target proteins were detected by rat anti F4/80 (1:500, Biolegend,San Diego, Calif.), rat anti-CD68 [FA-11] (1:200 abcam, Cambridge, UK)mouse anti GAPDH (1:10,000, abcam, Cambridge, UK), each in TBSTsupplemented with 5% skim milk powder (Sigma Aldrich, Munich, GER).Fluorescent conjugate secondary antibodies were applied for thedetection with a Li-Cor Odyssey imaging system (Bad Homburg, GER).

Histopathological Evaluation

After removal, organs were fixed in formalin for 24 h and transferredinto 70% ethanol. To ensure a non-biased comparison the same parts oforgans were analysed by HISTALIM (Montpellier, France). The samples wereprocessed on the Peloris automaton (Leica, Wetzlar, Germany) accordingto the 4 h program validated for mouse organs. The samples were embeddedin paraffin wax according to HISTALIM procedures. For the liver, spleenand femur samples, a section (3-5 μm thickness) was prepared anddeposited preferentially on Superfrost+slide (to ensure tissue adhesion)to be stained according to a validated hematoxylin/eosin (H&E) protocol.All the slides were digitalised with the Nanozoomer scanner (HamamatsuPhotonics, Hamamatsu, Japan) in conditions bright field conditions, withthe objective ×20, without Z stack. Slides were examined by a seniorhistopathologist to assess the toxicological effects of lyso-Gb1 on thetested mice.

Immunohistochemistry

For immunohistochemistry 5 μm paraffin sections of the spleens of 8-weektreated mice were subjected to rat anti-CD68 clone FA-11 (1:100, Bio-RadLaboratories, Raleigh, N.C., USA) and rat anti-mouse F4/80 Cl:A3-1(1:100, BioLegend, San Diego, Calif., USA). Sections were thendeparaffinised, rehydrated and pre-treated in the microwaves in 0.1 Mcitrate buffer (5 min 850 W and 5 min 340 W) followed by consecutiveincubation with 3% H₂O₂ in PBS to block endogenous peroxidases for 30min, then 3% bovine serum albumin with 1.5% normal goat serum (NGS) inPBS for 1 h to block nonspecific epitopes. Subsequently, sections wereexposed to the primary antibody in 3% NGS/PBS overnight at 4° C.Depending on the primary antibody and after washing in PBS, the sectionswere sequentially incubated for 1 h with the secondary anti-rat oranti-mouse IgG (1:200; Vector, Burlingame, Calif., USA),streptavidin-biotin-complex (ABC) reagent for 1 h (Vectastain-Elite;Vector, Burlingame, Calif., USA) and then finally visualised with3,-3,-diaminobenzidine (DAB, Sigma, Munich, Germany), which wasactivated with H2O2. Sections were counterstained with hematoxylin,dehydrated, mounted with DePeX and coverslipped.

Data Analysis

Visualisation and statistical data evaluation were carried out usingGraphPad Prism 5. Results are presented as median and range. Thenon-parametric two-tailed Mann-Whitney test was used to identifydifferences between treatment groups: hemoglobin, Hct and liver/spleenweight of control versus lyso-Gb1 treated mice at each indicated timepoint. Results were considered to be statistically significant for Pvalues <0.05*, <0.01**, <0.005***.

Glucocerebrosidase Enzyme Activity Measurement

Fresh-frozen liver and spleen samples were homogenized in pH 4.5adjusted ice-cold potassium-phosphate buffer (100 μL/mg tissue)supplemented with 0.15% Triton X-100 and 0.125% sodium taurocholate. Thetissue suspension was forced 10 times through a 22-gauge needle,equipped with a 2 mL syringe to release cell association. Thereafter,the suspension was subjected to 5 freeze/thaw cycles followed bycentrifugation at 15,000×g for 15 minutes at 4° C. to obtain a clearedlysate. The protein concentration of the GCase containing extracts wasmeasured and 9 μg whole protein was used for the enzymatic reactionusing 2 mM final concentration 4-Methylumbelliferyl-β-D-glucopyranoside(4-MUG) as substrate. The reaction was terminated by the addition of 0.2mL of 1.0 M glycine buffer (pH 10.5). The free fluorophore 4-MU wasdetermined in a microplate reader (Tecan, Minnedorf, Switzerland).

Example 2: Lyso-Gb1 Levels Upon Continuous Subcutaneous Administrationof Lyso-Gb1 to Mice

Male C57BL/6JRj mice were equipped with sub-cutaneous osmotic mini-pumpsin the back of their necks to achieve a long-term administration oflyso-Gb1 (12 weeks). Recurrent DBS sampling was carried out in order toinvestigate the lyso-Gb1 level development in the organ system of themice.

The result of the monitoring of blood lyso-Gb1 level in treated mice areshown in FIGS. 1 A and B.

As may be taken from FIGS. 1 A and B lyso-Gb1 levels were stronglyelevated already 24 hrs after treatment start (data not shown). Thelyso-Gb1 values observed after 24 hrs manifested throughout thetreatment phase. After 4, 8 and 12 weeks lyso-Gb1 level ranged between700-900 ng/ml, reflecting a more than 500-fold increase compared to thevehicle-treated mice. Vehicle-treated (or pre-dose) animals had lower,but detectable lyso-Gb1 values around 1.2-1.5 ng/ml blood (FIGS. 1 A andB).

After 4 weeks from treatment start the organs of the mice were subjectedto lyso-Gb1 analysis. The results are shown in FIG. 2A.

As may be taken from FIG. 2A, after 4 weeks from treatment (10mg*kg⁻¹*day⁻¹) start the heart, kidney, liver, spleen and brain wereanalysed for lyso-Gb1 accumulation. All peripheral organs of treatedmice showed strongly elevated levels of lyso-Gb1 compared to controlanimals. Highest levels of lyso-Gb1 were detected in the kidney whichindicates a predominant urinary elimination of the water-solublelyso-Gb1. This was verified by urinary levels of lyso-Gb1 (FIG. 2B). Adramatic increase of lyso-Gb1 was also observed in all other peripheralorgans whereas the fold change was more pronounced in liver and lesspronounced in spleen. A small, 2-fold increase of brain lyso-Gb1 wasattributed to carryover of blood in the tissue capillaries, which hadnot been cleared by perfusion prior to analysis.

Example 3: Analysis for Systemic Damage of Lyso-Gb1 Treated Mice

3.1 It was tested whether the high lyso-Gb1 levels caused systemicdamage to the animals. Such systemic damage was assessed by blood countanalysis. The results are shown in FIG. 3.

As evident from FIG. 3, left diagram, Hb values were slightly reducedand laid 2×SD below the baseline value at each time point indicating amild anaemia as defined by Raabe et al. (Raabe et al., 2011, supra). Thecontrol groups at all time points were compared and no statisticaldifference was found between them

More specifically, the animal displaced lowered Hct and Hct valuesdiffered at 4 and 8 weeks after treatment initiation (FIG. 3, rightdiagram). The depression of control values at week 12 was significantand could be a consequence of the vehicle (50% DMSO/50% propyleneglycol) treatment, because propylene glycol (ADDENDUM for PROPYLENEGLYCOL Supplement to the 1997 Toxicological Profile for PropyleneGlycol;https://www.atsdr.cdc.gov/toxprofiles/propylene_glycol_addendum.pdf) hasbeen demonstrated to be a hematologically effective compound. The lowinfusion volume, and reported tolerability of the compound in markedlyhigher doses (Thackaberry et al., 2010, Toxicol Sci, 117: 485-492),makes this unlikely. More important, however, is the fact that there isno further progression in Hct reduction in the lyso-Gb1 treated animals.

It is to be noted that no thrombocytopenia or abnormalities in whiteblood cells (WBC) were observed. Tables S1A and S1B (FIG. 6) summarizeall relevant parameters compiled in this study.

3.2 As anemia is a frequent result of enlarged spleen, another hallmarkof Gaucher disease, an organ weight analysis, a Western blot analysisand size and color analysis of the spleen was carried out. The resultsthereof are shown in FIG. 4A, FIG. 4B and FIG. 4C.

FIG. 4A summarizes the results obtained from organ weight analysis oflyso-Gb1 treated C57/BL/6JRj mice. As evident from FIG. 4A, the spleensof the treated mice were enlarged after 8 and 12 weeks from treatmentinitiation compared to the control organs. Liver weight was notstatistically distinct from control animals and the animals had thus nohepatomegaly.

In order to investigate the molecular changes of both spleen and liverin terms of tissue inflammation macrophagic marker proteins F4/80 andCD68 were analysed. FIG. 4B shows the result of a Western blot analysisof said inflammatory tissue markers F4/80 and CD68. Consistent withorgan weights, the levels of both proteins were elevated in the spleen,whereas only mild changes were observed in the liver.

Finally, visual inspection of half of a spleen from a control and alyso-Gb1 treated animal was performed. The organ samples illustrate thechange in size and a distinct colour appearance (see FIG. 4C).

A general histological stain of the spleens (also liver and femur) forall animals using H&E did not reveal significant abnormalities betweenvehicle and lyso-Gb1 treated organs (data not shown), butimmunohistochemical analysis using specific antibodies confirmed theWestern blot results. In detail, H&E-stained sections of spleen showed asimilar morphology in white and red pulp in both vehicle and lyso-Gb1treated mice (FIG. 5A, B). Immunohistochemical data revealed a strongincrease in CD68-immunoreactivity in lyso-Gb1 treated mice, whencompared to vehicle controls (FIG. 5C, D). In particular, accumulationof CD68-positive cells was seen near the capsule (FIG. 5E, F). Also,F4/80 immunoreactivity was increased in lyso-Gb1 treated mice comparedto vehicle controls (FIG. 5G, H). Higher magnification showed that thequalitative staining intensity of F4/80-positive cells was weak tomedium compared to that of CD68 positive cells in the spleen of lyso-Gb1treated mice (FIG. 5E, F to I, K).

Lyso-Gb1 is a potential substrate of GCase, which poses the questionwhether the treatment can influence proper enzyme function in theanimals as assessed before (Vacaro et al., 1985, Eur J Biochem 14:351-21). Enzyme measurements in spleen and liver lysates showed normalex vivo activity (FIG. 7) providing evidence that lyso-Gb1 did notsignificantly influence wild type GCase in the mice. This discovery isfurther supported by the fact that glucosylceramide levels are onlymoderately affected (FIG. 8).

Example 4: Interpretation of the Results of Examples 2 and 3

Ten weeks old C57BL/6JRj mice were subcutaneously treated with highdoses (10 mg*kg-1*day-1) of lyso-Gb1 for a period of up to 84 days (12weeks).

The mice developed a phenotype in blood and spleen comparable to a mildform of Gaucher disease, whereby phenotype resembles the phenotypeobserved in genetic mouse models of Gaucher disease type 1.

The treatment led to an accumulation of lyso-Gb1 in all major tissuesand a robust blood concentration of >500 ng/ml blood 4 weeks after beginof treatment, despite the presence of normal GCase. One explanation forthis could be the lower capacity of GCase to hydrolyse lyso-Gb1 incomparison to Glucosylceramide (Vacaro et al, supra). Typically, Gaucherpatient plasma lyso-Gb1 levels range from 50-250 ng/ml prior to enzymereplacement therapy (abbr. ERT) (Rolfs et al., PLoS One. 8(11): e79732)suggesting that the obtained concentration in the mouse circulation wascomparable, because, at least in humans, lyso-Gb1 blood level exceedsthe plasma values by a factor of >2 (unpublished data). Since it wasdiscovered that lyso-Gb1 levels are associated with disease severity(Dekker et al., 2011, supra; Rolfs et al., 2013, supra) it can thereforebe expected that the observed lyso-Gb1 concentration, if initiallycausative to GD symptom onset, was high enough to produce a similarphenotype in mice as in patients, most typically hepatosplenomegaly,anemia and bone disease.

Glucosylceramide and glucosylsphingosine are believed to be responsiblefor macrophagic organ infiltration and, consequently, the development oforganomegalies. Therefore, organ weight analysis of the treated animalswas performed. The gain in spleen weight was consistent with thedramatic increase in lyso-Gb1 in this organ and the observed elevationof CD68 and F4/80 antigen confirmed by Western blot andimmohistochemistry. This could suggest either an early stage or chronicinflammation within this tissue due to an increased number of immunecells, likely macrophages (Boven et al., 2004, Am J Clin Pathol 122:359-369; Kinoshita et al., 2010, J Hepatol, 53: 903-910), despite thevariation in cell populations positively stained for CD68 and F4/80. Incontrast, heart, lung and kidney appeared to be normal in size. Liverweight was slightly elevated at 8 weeks of treatment, albeit notstatistically significantly so, and histological examination of liversections suggested no pathophysiological condition. However,corresponding hepatic CD68 and F4/80 levels appeared mildly elevated,and we marked enlargement of the liver was observed in a subsequentexperiment where lyso-Gb1 treatment was started earlier (P20) (data notshown). This experiment also confirmed the spleen enlargement anddecreased Hb and Hct originally reported in genetic non-neuronopathic GDmice (Enquist et al., 2006, Proc Natl Acad Sci USA, 103: 13819-13824).

No further progression in phenotype severity was observed after 8 weeksof treatment with regards to organ weight and blood parameters; thissuggests a physiological adaptation by the mice, or perhaps the absenceof critical lyso-Gb1 concentrations at crucial target locations.Furthermore, the mice displayed no obvious health problems or functionalconstraints and did not lose weight throughout the experimental phase.This finding is contrary to the situation in a genetic mouse model withno apparent CNS involvement which demonstrated weight loss of 15%compared to healthy mice at 50 days of age, but no further progressivedecline (Mizukami et al., 2002, J Clin Invest. 109(9):1215-21.). Themice introduced by Mizukami et al. showed minimal glucosylceramidestorage and the absence of classic Gaucher tissue infiltration. However,their mice did display multisystemic inflammation reflecting elevatedhepatic TNF-α and IL-1β expression, highlighting that ratherinflammation is the key feature of GD that might not necessarily betypified by the presence of Gaucher cells. Our lyso-Gb1-treated miceshowed slight elevation of TNF-α and IL-1β in the blood, as observed byProcartaPlex® Multiplex Immunoassay, possibly indicating B-cellproliferation. However, we did not find significant upregulation of mostinvestigated cytokines (FIG. 6).

Late start of attendance of the mice (10 week old animals) is a likelyexplanation why the observed physiological changes in the peripheraltissues do not show the entire symptom spectrum of the disease. Earlierreports substantiated an elevation of lyso-Gb1 in the mouse body inembryonic phase (Orvisky et al., 2000, Pediatr Res. 48(2):233-7).However, this finding concerned a neuronopathic Gaucher mouse model. Todate, there is no pre-natal study on lyso-Gb1 in a non-neuronopathicmouse model, but it is reasonable to assume that lyso-Gb1 is alsoelevated during early developmental stages since it was found to beelevated in tissue of very young individuals (Orvisky et al., 2002, MolGenet Metab. 76(4):262-70).

In contrast to the GD patients and also the genetic mouse model of GDexamined by Orvisky and colleagues (Orvisky et al., 2000, supra), wherehighest lyso-Gb1 levels were observed in the spleen, we observed adifferent distribution, with the highest lyso-Gb1 levels measured in thekidney and liver. The reported finding from Orvisky and colleagues wouldindicate that lyso-Gb1 in GD originates from intra-splenic macrophagesources, whereas in our model, kidney and liver, not surprisingly,appeared to take up more of the subcutaneously administered lyso-Gb1.The negligible increase in lyso-Gb1 levels in the animal brain indicatesthat lyso-Gb1 is not able to cross the blood brain barrier. No CNSmeasurements were performed as the animals displayed no behavioralabnormalities, and the basal lyso-Gb1 level in the brain was higher thanin peripheral organs. This suggests better tolerance for lyso-Gb1 in theCNS.

Most known GD models have the disadvantage of rapid decline, whichhampers the close examination of disease progression. Most GD patientscarry the N370S allele, which is associated with a late disease onset,mild symptoms and slow disease progression. It was shown that certain,primarily homozygous, patients remain asymptomatic throughout life(Balwani et al., 2010, Arch Intern Med, 170: 1463-1469). Investigatingthe relationship between the pathophysiology of GD and the phenotype inthe lyso-Gb1 treated animals can be used to gain a better understandingof common and milder GD alleles. The N370S mutation can, however, alsobe found in patients with an early disease onset (Balwani et al, supra).Recent findings strongly suggest a critical role for the geneticbackground (Klein et al., 2016, Cell Rep, 16: 2546-53) in the phenotypicseverity of GD animal models and patients. It can also be speculatedthat differences in the metabolic pathways of the animals contribute todisease development. Consequently, the lyso-Gb1 treatment of a singleinbred mouse strain does not adequately reflect the complex spectrum ofphenotypes manifested inhuman GD.

The present mouse model could be used to investigate the efficacy oflyso-Gb1 neutralizing agents (e.g. antibodies and aptamers) as apotential drug therapy for GD. Engineering of monoclonal antibodies totarget hematological diseases, such as specific cancers, is becoming astandard therapy. Strategies to inactivate bioactive lipids byantibodies and aptamers have already been adopted (Sabbadini R A.;British Journal of Pharmacology. 2011; 162(6):1225-1238; WO 2011/15341;Purschke et al., Biochem J. 2014 Aug. 15; 462(1):153-62; internationalpatent application WO 2011/131371). The analysis of anti-inflammatorydrug effects on the visceral signs of GD could help to reveal whetherthe observed inflammatory tissue reaction is sufficient to protect themice from emerging disease signs, explaining their unaltered overallhealth status.

Example 5: Lyso-Gb1 in Parkinson's Disease

Patients suffering from Parkinson's disease having a mutation in GBA(glucocerebrosidase) show in increased level of lyso-Gb1 compared topatients suffering from Parkinson's disease without GBA mutation. Thelyso-Gb1 level is always below 12 ng/ml which is the critical thresholdfor patients suffering from Gaucher disease, but is still increased (7.9ng/ml vs. 2.8 ng/ml). The lyso-Gb1 level of these patients sufferingfrom Parkinson's disease is also increased compared to patients beingcarrier for Gaucher disease, but not being carrier for Parkinson'sdisease.

These data are supportive that lyso-Gb1 is involved in thepathophysiology of Parkinson's disease caused by mutations of GBA.

The features of the present invention disclosed in the specification,the claims, the sequence listing and/or the drawings may both separatelyand in any combination thereof be material for realizing the inventionin various forms thereof.

1.-15. (canceled)
 16. A method of using lyso-Gb1 as a druggable targetin the development of a drug antagonizing lyso-Gb1 by physicalinteraction for the treatment and/or prevention of Parkinson's disease,wherein the method comprises: determining in vitro whether a drugcandidate binds to lyso-Gb1 present outside of a cell, tissue or organ,determining in vivo whether the drug candidate is antagonizing theaction of lyso-Gb1 resulting in ameliorating at least one symptom of thedisease; wherein the drug candidate is a drug for the treatment and/orprevention of Parkinson's disease if the at least one symptom isameliorated, and wherein lyso-Gb1 is of formula (I)


17. The method of claim 16 further comprising determining the bindingaffinity of the drug candidate to lyso-Gb1.
 18. The method of claim 16,wherein the drug candidate is identified in a screening process from alibrary of compounds, wherein the screening process comprises providinga library of compounds and identifying from the library of compounds oneor more drug candidates capable of binding to lyso-Gb1.
 19. The methodof claim 16, wherein Parkinson's disease is caused by a mutation ofglucocerebrosidase.
 20. The method of claim 16, wherein Parkinson'sdisease is selected from the group consisting of a mild form of Gaucherdisease, type 1 Gaucher disease and non-neuronopathic Gaucher disease.21. The method of claim 16, wherein the at least one symptom is acentral nervous symptom.
 22. The method of claim 16, wherein the atleast one symptom is selected from the group consisting of ataxia,dementia, ocular apraxia, and parkinsonism.